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Experimental studies on the influence of male alcoholism on fetal development
ORIGINAL ARTICLES
Experimental Studies on the Influence of Male Alcoholism on Fetal Development Harumi Tanaka, MD, Nobuyuki Suzuki, MD and Masataka Arima, MD To study the effect of paternal chronic ethanol consumption on fetal development, an experimental rat model was established and compared to the fetal alcohol syndrome. Male and female Wistar rats were divided into 30% ethanol (E) and control groups. Before mating and during pregnancy the ethanol group and control group received E and water, respectively. Pregnancies were terminated on gestational day 21. The body weight, liver weight, blood glucose, serum insulin and cerebral CNPase activity were decreased in alcoholic males. The adverse effect of maternal chronic ethanol on fetal development was shown clearly and was not related to paternal ethanol. The adverse effect of paternal alcoholism on the fetus was shown in decreased litter size, or decreased body weight, cerebral weight and cerebral DNA, RNA and leucine incorporation into protein without a decrease in the litter size. The former finding was observed in the fetuses of aged male and female rats and latter in the fetuses of young female rats. In conclusion, both observations in our study indicate the adverse effect of paternal alcoholism on the fetal development. Tanaka H, Suzuki N, Arima M. Experimental studies on the influence of male alcoholism on fetal development. Brainpev 1982;4:1-6
Although sterility and/or impotence in the male are manifestations of chronic alcohol abuse that have been observed in man for many years, little has been done with respect to the effect of ethanol on spermatozoa and their ability to fertilize ova. In recent years there have been several experimental studies on development of spermatogenesis by alcohol [1-3]. Spermatogenesis adversely affected by alcohol possibly results in abnormal gametogenesis and abnormal
intrauterine development. After the description of the "fetal alcohol syndrome" showing developmental delay, and resulting from maternal alcoholism prior to and during gestation [4, 5] , a possible adverse influence of paternal alcoholism on the development of the offspring was suspected. The present study therefore examined the possible influence of male alcoholism on fetal development in rats. Materials and Methods
From the Division of Child Neurology, National Center for Nervous, Mental and Muscular Disorders, Kodaira, Tokyo. Received for publication: July 9, 1981. Accepted for pUblication: September 2, 1981.
Key words: Paternal alcoholism, fetal development, fetal alcohol syndrome, litter size. CQrrespondence address: Dr. Harumi Tanaka, Division of Child Neurology, National Center for Nervous, Mental and Muscular Disorders, 2620 Ogawahigashimachi, Kodaira, Tokyo 187, Japan.
Twenty-eight male and 61 virgin female albino rats of the Wistar strain were divided into ethanol and control groups. During the experimental period, the ethanol group and control group received 30% ethanol diluted with drinking water (vol/vol) (E) and water (W), respectively. Both groups were placed on a solid diet (F-2, Funabashi Corp., Chiba, 4.14 kcaljg) ad lib. According to the conditions of male and fe-
Table 1 Materials and methods (Exp 1) Premating period
Mating period
Gest. period
o
rWl
W~W
E-W W
E-E
W-E
Exp A male: 9w -lOOd female: 7w -lOOd Exp B male: lOw -101 d 8w -137d female: 8w -125d
600 9
2ld
rEl
W - W: male-female W : water E : 30% ethanol See text for details.
Table 2 Materials and methods (Exp 2) Premating Period
Mating Period
Gest. Period
o
66d W - W 102d W - W ly2mW-W
W1
66d E-W 102d E-W ly2mE-W
~Eorw-1
Male 66d: 102d: ly 2m: Female:
10w- 66d 8w-102d 9w-ly 2m lOw
r--W
2ld
W-
W - W: male-female W : water E : 30% ethanol See text for details.
male, two experiments were performed. Exp 1 as shown in Table 1 involved 4 kinds of matings between males and females. Before mating both male and female were pretreated with E or W. In Exp A, male rats at 9 weeks of age and female rats at 7 weeks of age were pretreated for 100 days. In Exp B, male rats at 10 weeks of age or 8 weeks of age, and female rats at 8 weeks of age were pretreated for 101, 137 and 125 days, respectively. During mating each male rat was placed in a cage with 2 females and all were given the solid diet and W ad lib. After successful matings confirmed by the presence of spermatozoa in vaginal smears, pregnant rats were given the same diet as that during the premating period. In another experiment (Exp 2), shown in Table 2, female rats at 10 weeks of age without 2 Brain & Development, Vol 4, No 1, 1982
300
•
•
o~o----------~~----------~~--~
days after bi rth
Fig 1 Effect of chronic ethanol administration on male body weight. Each point and bar represent the mean ± SD for control males . • shows ethanol males. Only statistically significant differences from respective controls are indicated; *** p < 0.01, ** p < 0.02, * p < 0.05.
pretreatment were mated with 3 kinds of male rats; beginning age of pretreatment at 10 weeks, 8 weeks and 9 weeks, and pretreatment period for 66 days, 102 days and 1 year 2 months, respectively. All pregnancies were terminated on gestational day 21 by Cesarean section. All parents and fetuses were examined for weight, litter size and gross abnormalities. Blood glucose, blood ethanol, serum insulin, DNA, RNA, protein, 2' 3' cyclic nucleotide 3' -phosphohydrolase (CNPase), 14C-leucine incorporation into cerebral protein in vitro and 14C orotic acid incorporation into cerebral RNA in vivo were also measured. Mean significance was calculated according to'the "Student's" t test. Results Paternal Aspects The male alcoholic rats showed signs of intoxication from the 2nd to 3rd week of treatment with the animals being less excitable and exhibiting less resistance to handling than the control group. Throughout the course of the investigation the control rats showed considerable weight gain, however, rats of the ethanol group lost weight and became lethargic. The body weights during the beginning of investigation in both groups are shown in Fig 1. As the ethanol groups showed a general increase at a slow rate after a transient decrease, the body weight of paternal rats at the time of mating was not lower than that at the beginning of treatment. Several paternal factors in the ethanol group
Table 3 Paternal factors
J"u/ml
Control Ethanol (% of control) 1. Body weight (g) Blood glucose (mg/dl) Brain weight: total (g) : cerebral (g) CNPase (U/mg protein) DNA (p.g/g wet weight) (J.Lg/mg protein) RNA (p.g/g wet weight) (p.g/mg protein) Liver weight (g) DNA (p.g/g wet weight) (p.g/mg protein) RNA (p.g/g wet weight) (p.g/mg protein)
2. Body weight (g) Mean testis weight (g)
437
328
( 75)
61
40
( 66)
1.94 1.39 3.47 1.19 7.39 1.97 12.2 12.72 2.55 10.7 10.4 43.4
1.93 1.35 2.82 1.06 7.55 1.93 13.7 9.40 3.12 13.8 10.6 46.9
( 99) ( 97) ( 81)
( 89) (102) ( 98) (112) ( 74) (122) (129) (102) (108)
"
V> C
2 ClJ
V>
~~~~
::'----"1
mg/lOOml 240
f
-----I1L-1
200
-ClJ
ov>
co ow
52100 ClJOl
"0"0
00
~~
.0.0
12 months of exper illJent _ethanol
20
Dcontrol
Fig 2 Blood glucose, eihanol and insulin levels in
550 1.91
560 1.73
(102) ( 91)
were compared to the controls (Table 3). The male alcoholic rats treated for 2 months from 9 weeks after birth showed values slightly less than the control in body weight, blood glucose levels, CNPase and liver weight, as shown in part 1 of Table 3. Rats with the same body weight treated for 11 months from 8 weeks after birth showed almost the same levels of mean testis weight, as shown in part 2 of Table 3. Results for the blood glucose, ethanol and serum insulin levels are given in Fig 2. The alcoholic rats were hypoglycemic and hypoinsulinemic compared to the controls. Blood ethanol levels at 10 am to noon in 4 alcoholic rats were 124, 185,0 and 12 mg/dl, respectively. In the rat with an undetectable ethanol level, the blood glucose level was not much lower than that in the controls. Maternal and Fetal Aspects Table 4 summarizes the results of Exp 1. The adverse effect of maternal chronic ethanol intake on physical and biochemical findings of fetuses was shown clearly and was not related to paternal ethanol. These abnormalities are referred to as the "fetal alcohol syndrome." The influence of paternal alcoholism on fetal factors was demonstrated in the difference
male alcoholic rats. Blood glucose was determined by Dextrostix. Blood ethanol was measured spectrophotometrically with alcohol dehydrogenase and NAD. Serum insulin level was determined by a doubleantibody radioimmunoassay.
between WoW and E-W. In both Exp A and B, the mean number of fetuses per litter of females mated with control males had a tendency to be greater than that of females mated with alcoholic rats. Considering the mean litter size, the adverse effect of paternal alcoholism on the fetus was shown in decreased body weight, cerebral weight and leucine incorporation into cerebral protein without a decrease in the litter size in Exp A or a decreased litter size in Exp B. As both male .and female rats in Exp B were older than those in Exp A at the time of mating, these findings are explained by the small litter size due to resorption in the case of worse paternal conditions or by the decreased body weight and cerebral weight in the case of better conditions. Table 5 summarizes the results of Exp 2 in the fetuses of young female rats. Although no pregnancy from matings with control males was observed after 1 year 2 months of experimental period, one pregnancy from matings with alcoholic males was successful after the same experimental period. In this experiment the adverse effect of paternal alcoholism on the fetuses was shown in the significantly decreased mean body weight and cerebral DNA and RNA contents. Tanaka et al: Alcoholic male and fetus 3
Table 4' Influence of paternal and/or maternal alcoholism on maternal and fetal factors (gestational day 21) (Exp 1)
ExpA
Maternal body weight (g,d.O) (g) 279 ± 20 Litter size 14.0±1.7 Fetal body weight (g) 4.73 ± 0.30 Fetal cerebral weight (mg) 149 ± 10 Cerebral DNA (}.Ig) 827 ± 59 Cerebral RNA (}.Ig) 469 ± 41 Leucine incorporation into 25.9 ± 4.6 cerebral protein (dpm x 10 4 /cerebrum)
ExpB
W-E
E-E
236 ± 26 ( 3) 9.3 ± 2.1 ( 3) 3.09 ± 1.01 [26] 112±11 [21] 725 ± 42 [ 6] 382 ± 44 [ 6] 13.2 ± 2.9 ( 3)
223 ± 4 ( 3) ( 1) 11 3.78 ± 0.38 [11] 123 ± 11 [ 8] 766 ± 4 [ 2] 423 ± 2 [ 2] ( 1) 18.6
W-E
E-E
E-W
W-W ( 3) ( 3) [42] [24] [ 6] [ 6] ( 3)
*** *** **
277 10.7 4.39 132 822 471 11.8
± 11 ± 6.5 ± 0.77 ± 10 ± 43 ± 23 ± 2.9
( 3) ( 3) [32] [23] [ 6] [ 6] ( 3)
E-W
W-W
Maternal body weight (g.d.O) (g) 288 ± 11 (5) Litter size 14.0 ± 1.8 (4) Fetal body weight (g) 4.72 ± 0.44 [56] Fetal cerebral weight (mg) 131 ± 7 [29] Cerebral protein (mg) 8.82 ± 1.29 [18] Cerebral RNA (}.Ig) 541 ± 48 [18] Orotic acid incorporation into 12.0 ± 1.0 [ 9] cerebral RNA (% of whole homogenate for 4 hrs)
*
252 11.5 3.55 113 7.73 490 11.9
287 ± 19 (5) 8.0 ± 4.8 (5) 4.85 ± 0.66 [40] 129 ± 10 [16] 9.30 ± 1.40 [21] 557 ± 33 [21] 12.1 ± 1.1 [11]
Data expressed as mean ± SD (no of dams) or [no of fetuses]. Significant differences between W - W and E - W; *** P < 0.01, W - W: male-female, W: water, E: 30% ethanol.
± 25 ± 2.4 ± 0.65 ± 10 ± 0.88 ± 48 ± 1.4
(6) (6) [68] [26] [21] [21] [10]
251 9.4 3.81 113 8.42 516 10.6
± 21 (6) ± 1.5 (5) ± 0.83 [46] ± 13 [19] ± 1.28 [15] ± 61 [15] ± 1.2 [ 8]
** p < 0.02, * p < 0.10.
Table 5 Influence of paternal alcoholism on maternal and fetal factors (gestational day 21) (Exp 2) 66d
W-W
102d
E-W
W-W
Maternal body weight (g.d.O) (g)
207±16
Litter size
11.4±2.3 ( 5)
Fetal body weight (g)
5.88±0.25 [45] *** 5.66 ±0.42 [35]
Fetal cerebral weight (mg)
166±8
( 5)
213±11
11.3 ±1.0 ( 4)
[33]
Cerebral DNA (mg) l.1S±0.27[ 8]
( 4)
163±11
*
[29]
214±5
( 4)
9.5 ± 1.9 ( 4)
1y2m
E-W
E-W ( 5)
248
( 1)
10.8±2.8 ( 5)
17
( 1)
209±19
6.07 ±0.37 [30] *** 5.34 ±0.60 [45] 163±9
[19]
160±13
[37]
4.50±0.35 [17] 146±8
[11]
0.91±0.12[ 6]
0.92±0.04[ 6]
0.86±0.14[ 8]
L55±0.13[ 2]
CerebraIRNA(mg) 0.60±0.05[ 8] * 0.55±0.04[ 6] Leucine incorporationintocerebral 4.44±0.90[10] 4.38±0.69[ 8] protein (% of whole homogenate for 30 min)
0.59±0.06[ 6]
0.60±0.04[ 8]
0.56±0.04[ 2]
4.47±0.86[ {l]
4.78±1.18[1l]
4.58±0.34[ 3]
Data expressed as meant SD (no of dams) or [no of fetuses]. Significant differences between W - Wand E - W; *** P < 0.01, W - W: male-female, W: water, E: 30% ethanol.
4 Brain & Development, Vol 4, No 1,1982
* p < 0.10. .
Table 6 Influence of paternal alcoholism on fetal growth index'"
w-w
E-W
Significance
Exp 1 Exp A 0.663 ± 0.109 (3) 0.468 ± 0.218 (3) Exp B 0.662 ± 0.064 (4) 0.388 ± 0.192 (5) p Total Exp 2 66d 102d
0.662 ± 0.077 (7) 0.418 ± 0.191 (8) p
< 0.05 < 0.01
0.655 ± 0.138 (5) 0.636 ± 0.054 (4) 0.562 ± 0.122 (4) 0.584 ± 0.093 (5)
* Growth index =
Mean fetal weight x Litter size 100
Data expressed as mean ± SD (no of dams). W - W: male-female, W: water, E: 30% ethanol.
The mean fetal growth-index which takes litter size into consideration was significantly smaller in the ethanol males than in the controls in Exp 1, especially in Exp B (Table 6). On the other hand, the mean fetal growth-index from younger females in Exp 2 showed the same values in the ethanol groups as in the controls. Discussion Previous investigators have reported that the number of offspring per litter of females mated with controls was significantly greater than that of females mated with alcoholic rats [6,7] , and mice [8], but that the mean fetal weight was significantly greater in the ethanol groups than in the controls [6]. The ratio of the pups surviving to weaning was reduced in litters sired by ethanol-treated males as compared with controls in rats [7] and in mice [8]. In the present study the decreased litter size among matings of old females with alcoholic males was observed, confirming the previous observations. 'On the other hand, the observation of decreased body weight, cerebral weight and cerebral DNA, RNA and leucine incorporation without a decrease in the litter size among matings of rather younger females with alcoholic males has not been described before. Both observations in our study - small litter size or decreased fetal development with the alcoholic male-indicate the adverse effect of paternal alcoholism on the fetal development. In the male ethanol can be related to lower-
ed circulating levels of testosterone [6,9]. Previous investigators have equated the long-term consequences of male alcohol use to a twostep process by which an individual initially experiences a reversible, diminished rate. of testosterone synthesis and the accompanymg aspermatogenesis; however, as hep~tic and testicular damage progresses, the resultmg damage become essentially irreversible [10, 11]. Badr et al [12] reported that ethanol produces its most dramatic effect on the male fertility potential during the first 4 to 13 days after treatment withdrawal in the dominant lethal assay procedure. Dominant lethal mutations have been regarded as imposing no genetic hazards on man since they merely result in abortion. As a significantly higher correlation between the incidence of birth defects and the drinking behavior of the fathers is indicated [12] , it is of interest whether paterna! ethanol could produce a wider range of genetIc effects beside dominant lethals. In this study hypoglycemia and hypoinsulinemia in male alcoholic rats were observed. In rat males after chronic ethanol ingestion, liver glycogen levels and blood glucose content were rather decreased [13] , which may be explained by increased utilization of glucose by peripheral tissues. Hypoglycemia in our male rats corresponds to this previous observation. ~thanol also inhibits hepatic gluconeogenesIs [14], which is caused by the alcohol dehydrogenase reaction, decreasing the (free NAD+) / (free NADH) ratio. From these observations hypoinsulinemia in our male rats can not be explained. On the other hand, the significance of zinc deficiency "in chronic alcoholism has been reported [15]. The increased loss of body zinc may indicate the hypoinsulinemia. It is also suspected that abnormal spermatogenesis in alcoholic rats may be caused by the zinc deficiency. Rydelius [16] has reported in "children of alcoholic fathers, their social adjustment and their health status over 20 years" that the children especially boys, of alcoholic fathers accou~ted for a large number of visits to hospitals for treatment of somatic symptoms and psychiatric problems. In order to resolve the biological nature of the social signific~ce in children of alcoholic fathers, further studIes are clearly needed.
Tanaka et al: Alcoholic male and fetus 5
Acknowledgment This study was partly supported by Grant No 81-0107, from the National Center for Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare, Japan. References 1. Abel EL. A review of alcohol's effects on sex and reproduction. Drug Alcohol Depend 1980;5: 321-32. 2. Anderson RA, Reddy JM, Oswald C, Willis BR, Zaneveld LID. Decreased male fertility induced by chronic alcohol ingestion (abstract). Fed Proc 1980;39:542. 3. Anderson RA, Willis BR, Oswald C, Gupta A, Zaneveld LJD. Delayed male sexual maturation induced by chronic ethanol ingestion (abstract). Fed Proc 1981;40:825. 4. Jones KL, Smith DW, Ulleland CN, Streissguth AP. Pattern of malformation in offspring of chronic alcoholic mothers. Lancet 1973;1:126771. 5. Tanaka H, Arima M, Suzuki N. The fetal alcohol syndrome in Japan. Brain Dev (Tokyo) 1981;3: 305-11. 6. Klassen RW, Persaud TVN. Experimental studies on the influence of male alcoholism on pregnancy and progeny. Exp Pathol 1976;12:38-45. 7. Pfeifer WD, Mackinnon JR, Seiser RL. Adverse effects of paternal alcohol consumption on offspring in the rat (abstract). Bull Psychonomic
6 Brain & Development, Vol 4, No 1,1982
Soc 1977;10:246. 8. Anderson RA, Beyler SA, Zaneveld LID. Alterations of male reproduction induced by chronic ingestion of ethanol: development of an animal model. Ferti! Steril 1978;30:103-5. 9. Rachamin G, Macdonald JA, Wahid S, Clapp JJ, Khanna JM, Israel Y. Modulation of alcohol dehydrogenase and ethanol metabolism by sex hormones in the spontaneously hypertensive rat. Biochem J 1980;186:483-90. 10. Van Thiel DH, Lester R. Sex and alcohol. N Engl J Med 1974;291:251-3. 11. Murono EP, Lin T, Osterman J, Nankin HR. Direct inhibition of testosterone synthesis in rat testis interstitial cells by ethanol: possible sites of action. Steroids 1980;36:619-31. 12. Badr FM, Badr RS. Induction of dominant lethal mutation in male mice by ethyl alcohol. Nature 1975;253:134-6. 13. Winston GW, Reitz RC. Effects of chronic ethanol ingestion on glucose homeostasis in males and females. Life Sci 1980;26:201-9. 14. Krebs HA, Freedland RA, Hems R, Stubbs M. Inhibition of hepatic gluconeogenesis by ethanol. Biochem J 1969;112:117-24. 15. Helwic HL, Hoffer EM, Thielen WC et al. Urinary and serum zinc levels in chronic alcoholism. Am J Clin Pathol 1966;45:156-9. 16. Rydelius P-A. Children of alcoholic fathers. Their social adjustment and their health status over 20 years. Acta Paediatr Scand [Suppl 286] 1981:7-89.
Experimental Studies on the Influence of Male Alcoholism on Fetal Development Harumi Tanaka, MD, Nobuyuki Suzuki, MD and Masataka Arima, MD To study the effect of paternal chronic ethanol consumption on fetal development, an experimental rat model was established and compared to the fetal alcohol syndrome. Male and female Wistar rats were divided into 30% ethanol (E) and control groups. Before mating and during pregnancy the ethanol group and control group received E and water, respectively. Pregnancies were terminated on gestational day 21. The body weight, liver weight, blood glucose, serum insulin and cerebral CNPase activity were decreased in alcoholic males. The adverse effect of maternal chronic ethanol on fetal development was shown clearly and was not related to paternal ethanol. The adverse effect of paternal alcoholism on the fetus was shown in decreased litter size, or decreased body weight, cerebral weight and cerebral DNA, RNA and leucine incorporation into protein without a decrease in the litter size. The former finding was observed in the fetuses of aged male and female rats and latter in the fetuses of young female rats. In conclusion, both observations in our study indicate the adverse effect of paternal alcoholism on the fetal development. Tanaka H, Suzuki N, Arima M. Experimental studies on the influence of male alcoholism on fetal development. Brainpev 1982;4:1-6
Although sterility and/or impotence in the male are manifestations of chronic alcohol abuse that have been observed in man for many years, little has been done with respect to the effect of ethanol on spermatozoa and their ability to fertilize ova. In recent years there have been several experimental studies on development of spermatogenesis by alcohol [1-3]. Spermatogenesis adversely affected by alcohol possibly results in abnormal gametogenesis and abnormal
intrauterine development. After the description of the "fetal alcohol syndrome" showing developmental delay, and resulting from maternal alcoholism prior to and during gestation [4, 5] , a possible adverse influence of paternal alcoholism on the development of the offspring was suspected. The present study therefore examined the possible influence of male alcoholism on fetal development in rats. Materials and Methods
From the Division of Child Neurology, National Center for Nervous, Mental and Muscular Disorders, Kodaira, Tokyo. Received for publication: July 9, 1981. Accepted for pUblication: September 2, 1981.
Key words: Paternal alcoholism, fetal development, fetal alcohol syndrome, litter size. CQrrespondence address: Dr. Harumi Tanaka, Division of Child Neurology, National Center for Nervous, Mental and Muscular Disorders, 2620 Ogawahigashimachi, Kodaira, Tokyo 187, Japan.
Twenty-eight male and 61 virgin female albino rats of the Wistar strain were divided into ethanol and control groups. During the experimental period, the ethanol group and control group received 30% ethanol diluted with drinking water (vol/vol) (E) and water (W), respectively. Both groups were placed on a solid diet (F-2, Funabashi Corp., Chiba, 4.14 kcaljg) ad lib. According to the conditions of male and fe-
Table 1 Materials and methods (Exp 1) Premating period
Mating period
Gest. period
o
rWl
W~W
E-W W
E-E
W-E
Exp A male: 9w -lOOd female: 7w -lOOd Exp B male: lOw -101 d 8w -137d female: 8w -125d
600 9
2ld
rEl
W - W: male-female W : water E : 30% ethanol See text for details.
Table 2 Materials and methods (Exp 2) Premating Period
Mating Period
Gest. Period
o
66d W - W 102d W - W ly2mW-W
W1
66d E-W 102d E-W ly2mE-W
~Eorw-1
Male 66d: 102d: ly 2m: Female:
10w- 66d 8w-102d 9w-ly 2m lOw
r--W
2ld
W-
W - W: male-female W : water E : 30% ethanol See text for details.
male, two experiments were performed. Exp 1 as shown in Table 1 involved 4 kinds of matings between males and females. Before mating both male and female were pretreated with E or W. In Exp A, male rats at 9 weeks of age and female rats at 7 weeks of age were pretreated for 100 days. In Exp B, male rats at 10 weeks of age or 8 weeks of age, and female rats at 8 weeks of age were pretreated for 101, 137 and 125 days, respectively. During mating each male rat was placed in a cage with 2 females and all were given the solid diet and W ad lib. After successful matings confirmed by the presence of spermatozoa in vaginal smears, pregnant rats were given the same diet as that during the premating period. In another experiment (Exp 2), shown in Table 2, female rats at 10 weeks of age without 2 Brain & Development, Vol 4, No 1, 1982
300
•
•
o~o----------~~----------~~--~
days after bi rth
Fig 1 Effect of chronic ethanol administration on male body weight. Each point and bar represent the mean ± SD for control males . • shows ethanol males. Only statistically significant differences from respective controls are indicated; *** p < 0.01, ** p < 0.02, * p < 0.05.
pretreatment were mated with 3 kinds of male rats; beginning age of pretreatment at 10 weeks, 8 weeks and 9 weeks, and pretreatment period for 66 days, 102 days and 1 year 2 months, respectively. All pregnancies were terminated on gestational day 21 by Cesarean section. All parents and fetuses were examined for weight, litter size and gross abnormalities. Blood glucose, blood ethanol, serum insulin, DNA, RNA, protein, 2' 3' cyclic nucleotide 3' -phosphohydrolase (CNPase), 14C-leucine incorporation into cerebral protein in vitro and 14C orotic acid incorporation into cerebral RNA in vivo were also measured. Mean significance was calculated according to'the "Student's" t test. Results Paternal Aspects The male alcoholic rats showed signs of intoxication from the 2nd to 3rd week of treatment with the animals being less excitable and exhibiting less resistance to handling than the control group. Throughout the course of the investigation the control rats showed considerable weight gain, however, rats of the ethanol group lost weight and became lethargic. The body weights during the beginning of investigation in both groups are shown in Fig 1. As the ethanol groups showed a general increase at a slow rate after a transient decrease, the body weight of paternal rats at the time of mating was not lower than that at the beginning of treatment. Several paternal factors in the ethanol group
Table 3 Paternal factors
J"u/ml
Control Ethanol (% of control) 1. Body weight (g) Blood glucose (mg/dl) Brain weight: total (g) : cerebral (g) CNPase (U/mg protein) DNA (p.g/g wet weight) (J.Lg/mg protein) RNA (p.g/g wet weight) (p.g/mg protein) Liver weight (g) DNA (p.g/g wet weight) (p.g/mg protein) RNA (p.g/g wet weight) (p.g/mg protein)
2. Body weight (g) Mean testis weight (g)
437
328
( 75)
61
40
( 66)
1.94 1.39 3.47 1.19 7.39 1.97 12.2 12.72 2.55 10.7 10.4 43.4
1.93 1.35 2.82 1.06 7.55 1.93 13.7 9.40 3.12 13.8 10.6 46.9
( 99) ( 97) ( 81)
( 89) (102) ( 98) (112) ( 74) (122) (129) (102) (108)
"
V> C
2 ClJ
V>
~~~~
::'----"1
mg/lOOml 240
f
-----I1L-1
200
-ClJ
ov>
co ow
52100 ClJOl
"0"0
00
~~
.0.0
12 months of exper illJent _ethanol
20
Dcontrol
Fig 2 Blood glucose, eihanol and insulin levels in
550 1.91
560 1.73
(102) ( 91)
were compared to the controls (Table 3). The male alcoholic rats treated for 2 months from 9 weeks after birth showed values slightly less than the control in body weight, blood glucose levels, CNPase and liver weight, as shown in part 1 of Table 3. Rats with the same body weight treated for 11 months from 8 weeks after birth showed almost the same levels of mean testis weight, as shown in part 2 of Table 3. Results for the blood glucose, ethanol and serum insulin levels are given in Fig 2. The alcoholic rats were hypoglycemic and hypoinsulinemic compared to the controls. Blood ethanol levels at 10 am to noon in 4 alcoholic rats were 124, 185,0 and 12 mg/dl, respectively. In the rat with an undetectable ethanol level, the blood glucose level was not much lower than that in the controls. Maternal and Fetal Aspects Table 4 summarizes the results of Exp 1. The adverse effect of maternal chronic ethanol intake on physical and biochemical findings of fetuses was shown clearly and was not related to paternal ethanol. These abnormalities are referred to as the "fetal alcohol syndrome." The influence of paternal alcoholism on fetal factors was demonstrated in the difference
male alcoholic rats. Blood glucose was determined by Dextrostix. Blood ethanol was measured spectrophotometrically with alcohol dehydrogenase and NAD. Serum insulin level was determined by a doubleantibody radioimmunoassay.
between WoW and E-W. In both Exp A and B, the mean number of fetuses per litter of females mated with control males had a tendency to be greater than that of females mated with alcoholic rats. Considering the mean litter size, the adverse effect of paternal alcoholism on the fetus was shown in decreased body weight, cerebral weight and leucine incorporation into cerebral protein without a decrease in the litter size in Exp A or a decreased litter size in Exp B. As both male .and female rats in Exp B were older than those in Exp A at the time of mating, these findings are explained by the small litter size due to resorption in the case of worse paternal conditions or by the decreased body weight and cerebral weight in the case of better conditions. Table 5 summarizes the results of Exp 2 in the fetuses of young female rats. Although no pregnancy from matings with control males was observed after 1 year 2 months of experimental period, one pregnancy from matings with alcoholic males was successful after the same experimental period. In this experiment the adverse effect of paternal alcoholism on the fetuses was shown in the significantly decreased mean body weight and cerebral DNA and RNA contents. Tanaka et al: Alcoholic male and fetus 3
Table 4' Influence of paternal and/or maternal alcoholism on maternal and fetal factors (gestational day 21) (Exp 1)
ExpA
Maternal body weight (g,d.O) (g) 279 ± 20 Litter size 14.0±1.7 Fetal body weight (g) 4.73 ± 0.30 Fetal cerebral weight (mg) 149 ± 10 Cerebral DNA (}.Ig) 827 ± 59 Cerebral RNA (}.Ig) 469 ± 41 Leucine incorporation into 25.9 ± 4.6 cerebral protein (dpm x 10 4 /cerebrum)
ExpB
W-E
E-E
236 ± 26 ( 3) 9.3 ± 2.1 ( 3) 3.09 ± 1.01 [26] 112±11 [21] 725 ± 42 [ 6] 382 ± 44 [ 6] 13.2 ± 2.9 ( 3)
223 ± 4 ( 3) ( 1) 11 3.78 ± 0.38 [11] 123 ± 11 [ 8] 766 ± 4 [ 2] 423 ± 2 [ 2] ( 1) 18.6
W-E
E-E
E-W
W-W ( 3) ( 3) [42] [24] [ 6] [ 6] ( 3)
*** *** **
277 10.7 4.39 132 822 471 11.8
± 11 ± 6.5 ± 0.77 ± 10 ± 43 ± 23 ± 2.9
( 3) ( 3) [32] [23] [ 6] [ 6] ( 3)
E-W
W-W
Maternal body weight (g.d.O) (g) 288 ± 11 (5) Litter size 14.0 ± 1.8 (4) Fetal body weight (g) 4.72 ± 0.44 [56] Fetal cerebral weight (mg) 131 ± 7 [29] Cerebral protein (mg) 8.82 ± 1.29 [18] Cerebral RNA (}.Ig) 541 ± 48 [18] Orotic acid incorporation into 12.0 ± 1.0 [ 9] cerebral RNA (% of whole homogenate for 4 hrs)
*
252 11.5 3.55 113 7.73 490 11.9
287 ± 19 (5) 8.0 ± 4.8 (5) 4.85 ± 0.66 [40] 129 ± 10 [16] 9.30 ± 1.40 [21] 557 ± 33 [21] 12.1 ± 1.1 [11]
Data expressed as mean ± SD (no of dams) or [no of fetuses]. Significant differences between W - W and E - W; *** P < 0.01, W - W: male-female, W: water, E: 30% ethanol.
± 25 ± 2.4 ± 0.65 ± 10 ± 0.88 ± 48 ± 1.4
(6) (6) [68] [26] [21] [21] [10]
251 9.4 3.81 113 8.42 516 10.6
± 21 (6) ± 1.5 (5) ± 0.83 [46] ± 13 [19] ± 1.28 [15] ± 61 [15] ± 1.2 [ 8]
** p < 0.02, * p < 0.10.
Table 5 Influence of paternal alcoholism on maternal and fetal factors (gestational day 21) (Exp 2) 66d
W-W
102d
E-W
W-W
Maternal body weight (g.d.O) (g)
207±16
Litter size
11.4±2.3 ( 5)
Fetal body weight (g)
5.88±0.25 [45] *** 5.66 ±0.42 [35]
Fetal cerebral weight (mg)
166±8
( 5)
213±11
11.3 ±1.0 ( 4)
[33]
Cerebral DNA (mg) l.1S±0.27[ 8]
( 4)
163±11
*
[29]
214±5
( 4)
9.5 ± 1.9 ( 4)
1y2m
E-W
E-W ( 5)
248
( 1)
10.8±2.8 ( 5)
17
( 1)
209±19
6.07 ±0.37 [30] *** 5.34 ±0.60 [45] 163±9
[19]
160±13
[37]
4.50±0.35 [17] 146±8
[11]
0.91±0.12[ 6]
0.92±0.04[ 6]
0.86±0.14[ 8]
L55±0.13[ 2]
CerebraIRNA(mg) 0.60±0.05[ 8] * 0.55±0.04[ 6] Leucine incorporationintocerebral 4.44±0.90[10] 4.38±0.69[ 8] protein (% of whole homogenate for 30 min)
0.59±0.06[ 6]
0.60±0.04[ 8]
0.56±0.04[ 2]
4.47±0.86[ {l]
4.78±1.18[1l]
4.58±0.34[ 3]
Data expressed as meant SD (no of dams) or [no of fetuses]. Significant differences between W - Wand E - W; *** P < 0.01, W - W: male-female, W: water, E: 30% ethanol.
4 Brain & Development, Vol 4, No 1,1982
* p < 0.10. .
Table 6 Influence of paternal alcoholism on fetal growth index'"
w-w
E-W
Significance
Exp 1 Exp A 0.663 ± 0.109 (3) 0.468 ± 0.218 (3) Exp B 0.662 ± 0.064 (4) 0.388 ± 0.192 (5) p Total Exp 2 66d 102d
0.662 ± 0.077 (7) 0.418 ± 0.191 (8) p
< 0.05 < 0.01
0.655 ± 0.138 (5) 0.636 ± 0.054 (4) 0.562 ± 0.122 (4) 0.584 ± 0.093 (5)
* Growth index =
Mean fetal weight x Litter size 100
Data expressed as mean ± SD (no of dams). W - W: male-female, W: water, E: 30% ethanol.
The mean fetal growth-index which takes litter size into consideration was significantly smaller in the ethanol males than in the controls in Exp 1, especially in Exp B (Table 6). On the other hand, the mean fetal growth-index from younger females in Exp 2 showed the same values in the ethanol groups as in the controls. Discussion Previous investigators have reported that the number of offspring per litter of females mated with controls was significantly greater than that of females mated with alcoholic rats [6,7] , and mice [8], but that the mean fetal weight was significantly greater in the ethanol groups than in the controls [6]. The ratio of the pups surviving to weaning was reduced in litters sired by ethanol-treated males as compared with controls in rats [7] and in mice [8]. In the present study the decreased litter size among matings of old females with alcoholic males was observed, confirming the previous observations. 'On the other hand, the observation of decreased body weight, cerebral weight and cerebral DNA, RNA and leucine incorporation without a decrease in the litter size among matings of rather younger females with alcoholic males has not been described before. Both observations in our study - small litter size or decreased fetal development with the alcoholic male-indicate the adverse effect of paternal alcoholism on the fetal development. In the male ethanol can be related to lower-
ed circulating levels of testosterone [6,9]. Previous investigators have equated the long-term consequences of male alcohol use to a twostep process by which an individual initially experiences a reversible, diminished rate. of testosterone synthesis and the accompanymg aspermatogenesis; however, as hep~tic and testicular damage progresses, the resultmg damage become essentially irreversible [10, 11]. Badr et al [12] reported that ethanol produces its most dramatic effect on the male fertility potential during the first 4 to 13 days after treatment withdrawal in the dominant lethal assay procedure. Dominant lethal mutations have been regarded as imposing no genetic hazards on man since they merely result in abortion. As a significantly higher correlation between the incidence of birth defects and the drinking behavior of the fathers is indicated [12] , it is of interest whether paterna! ethanol could produce a wider range of genetIc effects beside dominant lethals. In this study hypoglycemia and hypoinsulinemia in male alcoholic rats were observed. In rat males after chronic ethanol ingestion, liver glycogen levels and blood glucose content were rather decreased [13] , which may be explained by increased utilization of glucose by peripheral tissues. Hypoglycemia in our male rats corresponds to this previous observation. ~thanol also inhibits hepatic gluconeogenesIs [14], which is caused by the alcohol dehydrogenase reaction, decreasing the (free NAD+) / (free NADH) ratio. From these observations hypoinsulinemia in our male rats can not be explained. On the other hand, the significance of zinc deficiency "in chronic alcoholism has been reported [15]. The increased loss of body zinc may indicate the hypoinsulinemia. It is also suspected that abnormal spermatogenesis in alcoholic rats may be caused by the zinc deficiency. Rydelius [16] has reported in "children of alcoholic fathers, their social adjustment and their health status over 20 years" that the children especially boys, of alcoholic fathers accou~ted for a large number of visits to hospitals for treatment of somatic symptoms and psychiatric problems. In order to resolve the biological nature of the social signific~ce in children of alcoholic fathers, further studIes are clearly needed.
Tanaka et al: Alcoholic male and fetus 5
Acknowledgment This study was partly supported by Grant No 81-0107, from the National Center for Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare, Japan. References 1. Abel EL. A review of alcohol's effects on sex and reproduction. Drug Alcohol Depend 1980;5: 321-32. 2. Anderson RA, Reddy JM, Oswald C, Willis BR, Zaneveld LID. Decreased male fertility induced by chronic alcohol ingestion (abstract). Fed Proc 1980;39:542. 3. Anderson RA, Willis BR, Oswald C, Gupta A, Zaneveld LJD. Delayed male sexual maturation induced by chronic ethanol ingestion (abstract). Fed Proc 1981;40:825. 4. Jones KL, Smith DW, Ulleland CN, Streissguth AP. Pattern of malformation in offspring of chronic alcoholic mothers. Lancet 1973;1:126771. 5. Tanaka H, Arima M, Suzuki N. The fetal alcohol syndrome in Japan. Brain Dev (Tokyo) 1981;3: 305-11. 6. Klassen RW, Persaud TVN. Experimental studies on the influence of male alcoholism on pregnancy and progeny. Exp Pathol 1976;12:38-45. 7. Pfeifer WD, Mackinnon JR, Seiser RL. Adverse effects of paternal alcohol consumption on offspring in the rat (abstract). Bull Psychonomic
6 Brain & Development, Vol 4, No 1,1982
Soc 1977;10:246. 8. Anderson RA, Beyler SA, Zaneveld LID. Alterations of male reproduction induced by chronic ingestion of ethanol: development of an animal model. Ferti! Steril 1978;30:103-5. 9. Rachamin G, Macdonald JA, Wahid S, Clapp JJ, Khanna JM, Israel Y. Modulation of alcohol dehydrogenase and ethanol metabolism by sex hormones in the spontaneously hypertensive rat. Biochem J 1980;186:483-90. 10. Van Thiel DH, Lester R. Sex and alcohol. N Engl J Med 1974;291:251-3. 11. Murono EP, Lin T, Osterman J, Nankin HR. Direct inhibition of testosterone synthesis in rat testis interstitial cells by ethanol: possible sites of action. Steroids 1980;36:619-31. 12. Badr FM, Badr RS. Induction of dominant lethal mutation in male mice by ethyl alcohol. Nature 1975;253:134-6. 13. Winston GW, Reitz RC. Effects of chronic ethanol ingestion on glucose homeostasis in males and females. Life Sci 1980;26:201-9. 14. Krebs HA, Freedland RA, Hems R, Stubbs M. Inhibition of hepatic gluconeogenesis by ethanol. Biochem J 1969;112:117-24. 15. Helwic HL, Hoffer EM, Thielen WC et al. Urinary and serum zinc levels in chronic alcoholism. Am J Clin Pathol 1966;45:156-9. 16. Rydelius P-A. Children of alcoholic fathers. Their social adjustment and their health status over 20 years. Acta Paediatr Scand [Suppl 286] 1981:7-89.