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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1983, 1987
; Cicero et al., 1994
). In females, chronic ethanol consumption impairs the reproductive cycle and alters ovarian function (Van Thiel et al., 1978
; Eskay et al., 1981
; Cebral et al., 1998a
). In women, there is an elevated risk of infertility with high alcohol consumption (Mello, 1988
), and even moderate alcohol use can affect fertility (Grodstein et al., 1994
). 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, 1980
). 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, 1988
). 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 a
,b
). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 maltosedextrin (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., 1997, 1998a
).
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, 1975) 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 1617 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 12 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, 1971) 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 1617 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.
Statistics
Group differences were examined by the 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In vitro fertilization
Evaluation at 5 h post-insemination.
Table 2 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).
|
|
|
Evaluation of embryo quality.
Figure 1 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).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1995). 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., 1998b
). 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., 1998b
). Eskay et al. (1981) suggested that ethanol in the range of 50100 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, 1986), before mating and through the preimplantation development (Sandor et al., 1980
) or exposure during culture in vitro of mouse embryos (Wiebold and Becker, 1987
; Kalmus and Buckenmaier, 1989
; Kowalczyk et al., 1996
). 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., 1988; Winston et al., 1991
). Retarded embryo development in the ethanol-treated females may be a consequence of augmented parthenogenetic development. Several authors (Kaufman, 1990
; Henery and Kaufman, 1992
) 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, 1990
). 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, 1984b
).
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., 1995).
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson, R. A., Willis, B. R., Oswald, C. and Zaneveld, L. J. (1983) Ethanol-induced male infertility: impairment of spermatozoa. Journal of Pharmacology and Experimental Therapeutics 225, 479486.[Abstract]
Anderson, R. A., Willis, B. R., Phillips, J. F., Oswald, C. and Zaneveld, L. J. (1987) Delayed pubertal development of the male reproductive tract associated with chronic ethanol ingestion. Biochemical Pharmacology 36, 21572167.[ISI][Medline]
Cebral, E., Lasserre, A., Rettori, V. and Gimeno, M. A. (1997) Impaired mouse fertilization by low chronic alcohol treatment. Alcohol and Alcoholism 32, 563572.[Abstract]
Cebral, E., Lasserre, A., Faletti, A. and Gimeno, M. A. (1998a) Response to ovulatory induction following moderate chronic ethanol administration in mice. Medical Science Research 26, 2931.[ISI]
Cebral, E., Lasserre, A., Motta, A. and Gimeno, M. A. (1998b) Mouse oocyte quality and prostaglandin synthesis by cumulus oocyte complex after moderate chronic ethanol intake. Prostaglandins, Leukotrienes and Essential Fatty Acids 58, 381387.[ISI][Medline]
Checiu, M. C. and Sandor, S. (1982) The effect of ethanol upon early development in mice and rats. IV. The effect of acute ethanol intoxication on day 4 of pregnancy upon implantation and early postimplantation development in mice. Morphology and Embryology 28, 127133.[Medline]
Checiu, M. and Sandor, S. (1986) The effect of ethanol upon early development in mice and rats. IX. Late effect of acute preimplantation intoxication in mice. Morphology and Embryology 32, 511.[Medline]
Chernoff, G. F. (1980) The fetal alcohol syndrome in mice: maternal variables. Teratology 22, 7175.[ISI][Medline]
Cicero, T. J., Nock, B., O'Connor, L., Adams, M. L., Sewing, B. N. and Meyer, E. R. (1994) Acute alcohol exposure markedly influences male fertility and fetal outcome in the male rat. Life Sciences 55, 901910.[ISI][Medline]
Dyban, A. P. and Khozai, L. I. (1980) Parthenogenetic development of ovulated mouse ova under influence of ethyl alcohol. Bulletin of Experimental Biology and Medicine 89, 528530.[ISI]
Eskay, R. L., Ryback, R. S., Goldman, M. and Majchrowicz, E. (1981) Effect of chronic ethanol administration on plasma levels of LH and the estrous cycle in the female rat. Alcoholism: Clinical and Experimental Research 5, 204206.[ISI][Medline]
Fraser, L. R. and Drury, L. M. (1975) The relationship between sperm concentration and fertilization in vitro of mouse eggs. Biology of Reproduction 13, 513518.[ISI][Medline]
Grodstein, F., Goldman, M. B. and Cramer, C. W. (1994). Infertility in women and moderate alcohol use. American Journal of Public Health 84, 14291432.[Abstract]
Henery, C. C. and Kaufman, M. H. (1992) Cleavage rate of haploid and diploid parthenogenetic mouse embryos during the preimplantation period. Molecular Reproduction and Development 31, 258263.[ISI][Medline]
Kalmus, G. W. and Buckenmaier, C. C. (1989) Effects of ethanol and acetaldehyde on cultured preimplantation mouse embryos. Experientia 45, 484487.[ISI][Medline]
Kaufman, M. H. (1985) An hypothesis regarding the origin of aneuploidy in man: indirect evidence from an experimental model. Journal of Medical Genetics 22, 171178.[Abstract]
Kaufman, M. H. (1990) Early Mammalian Development: Parthenogenetic Studies, pp. 144145. Cambridge University Press, Cambridge.
Kaufman, M. H. (1997) The teratogenic effects of alcohol following exposure during pregnancy, and its influence on the chromosome constitution of the pre-ovulatory egg. Alcohol and Alcoholism 32, 113128.[Abstract]
Kaufman, M. H. and Bain, I. M. (1984a) Influence of ethanol on chromosome segregation during the first and second meiotic divisions in the mouse egg. Journal of Experimental Zoology 230, 315320.[ISI][Medline]
Kaufman, M. H. and Bain, I. M. (1984b) The development potential of ethanol-induced monosomic and trisomic conceptus in the mouse. Journal of Experimental Zoology 231, 149155.[ISI][Medline]
Kaufman, M. H. and O'Neill, G. T. (1988) Aneuploidy induced by ethanol. In Aneuploidy, Part B, Induction and Test Systems, Vig, B. K. and Sandberg, A. A. eds, pp. 95122. Alan R. Liss, New York.
Kotch, L. E. and Sulik, K. K. (1992) Experimental fetal alcohol syndrome: proposed pathogenic basis for a variety of associated facial and brain anomalies. American Journal of Medical Genetics 44, 168176.[ISI][Medline]
Kowalczyk, C. L., Stachecki, J. J., Schultz, J. F., Leach, R. E. and Randall, C. A. (1996) Effects of alcohol on murine preimplantation development: relationship to relative membrane disordering potency. Alcoholism: Clinical and Experimental Research 20, 566571.[ISI][Medline]
Mello, N. K. (1988) Effects of alcohol abuse on reproductive function in women. In Recent Developments in Alcoholism, Galanter, M. ed., pp. 253276. Plenum, New York.
O'Neill, G. T. and Kaufman, M. H. (1989) Cytogenetic analysis of ethanol-induced parthenogenesis. Journal of Experimental Zoology 249, 182192.[ISI][Medline]
Pickering, S. J., Johnson, M. H., Braude, P. R. and Houliston, E. (1988) Cytoskeletal organization in fresh, aged and spontaneously activated human oocytes. Human Reproduction 3, 978989.[Abstract]
Randall, C. L. and Taylor, W. J. (1979) Prenatal ethanol exposure in mice: teratogenic effects. Teratology 19, 305312.[ISI][Medline]
Sandor, S., Checiu, M., Fazakas-Todea, I. and Garba, Z. (1980) The effect of ethanol upon early development in mice and rats. I. In vivo effect upon preimplantation and early postimplantation stages. Morphology and Embryology 26, 265274.
Takase, K., Ishikawa, M. and Hoshiai, H. (1995) Apoptosis on degeneration process of unfertilized mouse ova. Tohoku Journal of Experimental Medicine 175, 6976.[ISI][Medline]
Van Thiel, D. H., Gavaler, J., Lester, R. and Sherins, R. J. (1978) Alcohol-induced ovarian failure in the rat. Journal of Clinical Investigation 61, 624632.[ISI][Medline]
Weston, W. M., Greene, R. M., Uberti, M. and Pisano, M. M. (1994) Ethanol effects on embryonic craniofacial growth and development: implications for study of the fetal alcohol syndrome. Alcoholism: Clinical and Experimental Research 18, 177182.[ISI][Medline]
Whittingham, D. G. (1971) Culture of mouse ova. Journal of Reproduction and Fertility 14 (Suppl.), 721.
Wiebold, J. L. and Becker, W. C. (1987) In vivo and in vitro effects of ethanol on mouse preimplantation embryos. Journal of Reproduction and Fertility 80, 4957.[Abstract]
Winston, N., Johnson, M., Pickering, S. and Braude, P. (1991) Parthenogenetic activation and development of fresh and aged human oocytes. Fertility and Sterility 56, 904912.[ISI][Medline]