Alcohol,Vol. 7, pp. 21-25. ©Pergamon Press plc, 1990. Printed in the U.S.A.
0741-8329/90 $3.00 + .00
Actions of Ethanol on Hypothalamic and Pituitary Hormones in Prepubertal Female Rats W. L E S D E E S , *l C A R L W. S K E L L E Y , * J I L L K. H I N E Y * A N D C R A I G A. J O H N S T O N t
*Laboratory for Neuroendocrinology, Department of Anatomy, College of Veterinary Medicine Texas A&M University, College Station, TX 77843 and ~'College of Pharmacy, Pharmaceutical Sciences Unit Washington State University, Pullman, WA 99164 R e c e i v e d 30 June 1989; A c c e p t e d 23 A u g u s t 1989
DEES, W. L., C. W. SKELLEY, J. K. HINEY AND C. A. JOHNSTON. Actionsof ethanolon hypothalamicandpituitaryhormones in prepubertalfemale rats. ALCOHOL 7(1) 21-25, 1990.--To determine the effects of prepubertal ethanol (ETOH) exposure on hypothalamic and pituitary hormones known to be involved in the onset of female puberty, we have chronically exposed female rats to either a liquid-diet containing ETOH or an isocaloric control liquid-diet. An additional set of controls consisted of animals maintained on Lab Chow, and water provided ad lib. Our results indicate that the feeding regimen employed produced no differences with regard to body and reproductive organ weights, as well as any of the hormones measured between the two control groups. Conversely, ETOH-treated animals showed significantly lower body and reproductive organ weights than the control animals and although no differences were detected between ETOH-treated and control animals with regard to the hypothalamic content of somatostatin (SRIF), there was a significant increase in the hypothalamic content of growth hormone releasing hormone (GHRH), with a concomitant and significant decrease in the serum concentration of growth hormone (GH). Furthermore, the ETOH-treated animals showed a significant increase in the hypothalamic content of luteinizing hormone releasing hormone (LHRH) with a significant decrease in the serum concentration of luteinizing hormone (LH), but not follicle stimulating hormone (FSH). These results demonstrate for the first time that chronic, prepubertal ETOH administration alters the concentrations of specific hypothalamic and pituitary hormones which are known to be involved in the female pubertal process. Ethanol Prepubertal Growth hormone releasing hormone Somatostatin Luteinizing hormone releasing hormone Growth hormone Luteinizing hormone
THE effects of ETOH on the female pubertal process are poorly understood. Chronic ETOH administration has been shown to delay the onset of puberty in female rats, as evidenced by a delay in vaginal opening (VO), decreased uterine and ovarian weights and depressed ovarian function (5). By analyzing the female maturation process of both control and ETOH-treated rats through day 47, we recently have shown that the ETOH-treated rats remained in the anestrous phase of puberty and do not enter the early (first) proestrous phase of the peripubertal stage of development (7,10). However, the effects of prepubertal ETOH consumption on physiological parameters, such as possible brain and pituitary hormonal alterations during the late anestrous phase of puberty, by which ETOH may be blocking entry into the peripubertal period, still are not known. The present study was conducted to investigate possible neurotoxic effects of prepubertal ETOH exposure on specific hormones which are known to be extensively involved in the early pubertal process. Therefore, we have examined the chronic effects of ETOH in immature female rats just prior to entering the peripubertal period of development,
Follicle stimulating hormone
on the hypothalamic contents of growth hormone releasing hormone (GHRH), somatostatin (SRIF) and luteinizing hormone releasing hormone (LHRH), as well as the serum levels of growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH). METHOD
Animals and Surgery Immature female Sprague-Dawley rats of the Harlan line (Harlan, Inc., Houston, TX) were used. Each rat was anesthetized at 23 days of age with Equithesin (Fort Dodge Labs. Inc., Fort Dodge, IA) and surgically implanted with a permanent intragastric cannula by a procedure that we have described previously (11). The animals were then housed individually under controlled conditions of light (lights on, 0800 hr; lights off, 1800 hr) and temperature (23°C), with ad lib access to food (Purina Laboratory Chow) and tap water. Animals were allowed to recover from surgery for five days prior to the beginning of the experiment. In
~Requests for reprints should be addressed to W. Les Dees, Laboratory for Neuroendocrinology, Department of Anatomy, College of Veterinary Medicine, Texas A&M University, College Station, TX 77834.
21
DEES ET AL.
22
order to assess the effects of ETOH on specific hormones known to be involved in the early pubertal process, each rat was administered a specific diet regimen, then killed in the anestrous phase of puberty as described below.
Experimental Procedure Twenty-eight-day-old rats were weighed and assigned to one of three groups, each averaging between 60~65 g body weight. Group one consisted of 30 animals that received a 5% ETOH liquid-diet and group two consisted of 14 animals that received the isocaloric control liquid-diet (Bio-Serve, Inc., Frenchtown, NJ). Group three served as an additional set of controls which consisted of 15 animals that were cannulated and maintained on Lab Chow and water, ad lib, throughout the experiment. On day 29, the animals in groups I and II began receiving their respective liquid-diet regimen. These diets were administered in such a manner that 7.5 ml of the respective diet was injected via the intragastric cannula (5 injections of 1.5 ml each) equally dispersed over the lights-on period, then no more than 30 ml of diet was available ad lib (bottle) during the lights-off period. In order to provide adequate food supply for these immature growing animals, beginning on day 30 and again on days 32 and 34, the amount of diet injected via the cannula was increased by 0.5 ml per injection. Also, at these same times the amount of diet made available each night in the bottle was increased to 35, 40 and 50 ml, respectively. Thus, by day 34, each animal was receiving the maximum of 5 intragastric injections (3.0 ml each) of diet during the lights-on period and 50 ml of diet, ad lib, during the lights-off period. We have reported previously that this procedure enabled the liquid-diet control group to grow at exactly the same rate as the Lab Chow control group (7,10). Additionally, this method not only ensured that all liquid-diet fed animals received approximately the same number of calories per day, but also ensured that all of the ETOH-treated animals received the same amount of ETOH per day. Both the control and ETOH-treated animals were weighed and killed by decapitation between 0800 and 0900 hr at a mean of 34.0_+ 1.1 and 34.5_+ 1.3 days of age, respectively. The uterus and ovaries from each animal were removed and weighed to the nearest milligram and all animals were confirmed as being in the anestrous phase of puberty based on well-defined criteria which classify the different phases of the peripubertal period (1,15). Briefly, animals in the anestrous phase had closed vaginae, small uteri (up to 94 rag) and no intraluminal fluid. Trunk blood was collected, centrifuged at 3000 × g for 20 min, and the serum stored at - 20°C until assayed for GH, LH, and FSH. Brains were removed and hypothalami (with median eminence) excised by dissection using the pyriform lobes, caudal borders of the optic chiasm and mammillary bodies, and the thalamic adhesion as the lateral, rostral, caudal, and dorsal boundaries, respectively. The brain tissues were immediately frozen on dry ice and stored at - 7 0 ° C until processed further for assaying the hypothalamic contents of GHRH, SRIF and LHRH.
14ypothalamic Extracts and Peptide R1A's Each hypothalamus was lyophilized and subjected to acetic acid extraction for peptides by a procedure previously described (14). After the addition of 500 tzl of 2 N acetic acid, each sample was vortexed for one min and placed in a boiling water bath for five min to destroy protease. The homogenates were centrifuged at 4 5 0 0 × g for 30 min, and 300 ixl of supernatant fluid was removed and frozen. Samples were similarly extracted two more times (without boiling) with 500 Izl 0.1 N acetic acid, and 500 and
400 ixl, respectively, of supernatant fluid was removed after each centrifugation. The total supernatant fluid for each sample was lyophilized and stored at 4°C under desiccant. Each extract was reconstituted just before assay with 1 ml of phosphate-buffered saline pH 7.4. Extracts were measured for their GHRH content using a specific RIA. The antiserum used (RAS 8068) was raised against synthetic rat GHRH (rGHRH, Peninsula Labs, Belmont, CA) and does not cross-react (
0741-8329/90 $3.00 + .00
Actions of Ethanol on Hypothalamic and Pituitary Hormones in Prepubertal Female Rats W. L E S D E E S , *l C A R L W. S K E L L E Y , * J I L L K. H I N E Y * A N D C R A I G A. J O H N S T O N t
*Laboratory for Neuroendocrinology, Department of Anatomy, College of Veterinary Medicine Texas A&M University, College Station, TX 77843 and ~'College of Pharmacy, Pharmaceutical Sciences Unit Washington State University, Pullman, WA 99164 R e c e i v e d 30 June 1989; A c c e p t e d 23 A u g u s t 1989
DEES, W. L., C. W. SKELLEY, J. K. HINEY AND C. A. JOHNSTON. Actionsof ethanolon hypothalamicandpituitaryhormones in prepubertalfemale rats. ALCOHOL 7(1) 21-25, 1990.--To determine the effects of prepubertal ethanol (ETOH) exposure on hypothalamic and pituitary hormones known to be involved in the onset of female puberty, we have chronically exposed female rats to either a liquid-diet containing ETOH or an isocaloric control liquid-diet. An additional set of controls consisted of animals maintained on Lab Chow, and water provided ad lib. Our results indicate that the feeding regimen employed produced no differences with regard to body and reproductive organ weights, as well as any of the hormones measured between the two control groups. Conversely, ETOH-treated animals showed significantly lower body and reproductive organ weights than the control animals and although no differences were detected between ETOH-treated and control animals with regard to the hypothalamic content of somatostatin (SRIF), there was a significant increase in the hypothalamic content of growth hormone releasing hormone (GHRH), with a concomitant and significant decrease in the serum concentration of growth hormone (GH). Furthermore, the ETOH-treated animals showed a significant increase in the hypothalamic content of luteinizing hormone releasing hormone (LHRH) with a significant decrease in the serum concentration of luteinizing hormone (LH), but not follicle stimulating hormone (FSH). These results demonstrate for the first time that chronic, prepubertal ETOH administration alters the concentrations of specific hypothalamic and pituitary hormones which are known to be involved in the female pubertal process. Ethanol Prepubertal Growth hormone releasing hormone Somatostatin Luteinizing hormone releasing hormone Growth hormone Luteinizing hormone
THE effects of ETOH on the female pubertal process are poorly understood. Chronic ETOH administration has been shown to delay the onset of puberty in female rats, as evidenced by a delay in vaginal opening (VO), decreased uterine and ovarian weights and depressed ovarian function (5). By analyzing the female maturation process of both control and ETOH-treated rats through day 47, we recently have shown that the ETOH-treated rats remained in the anestrous phase of puberty and do not enter the early (first) proestrous phase of the peripubertal stage of development (7,10). However, the effects of prepubertal ETOH consumption on physiological parameters, such as possible brain and pituitary hormonal alterations during the late anestrous phase of puberty, by which ETOH may be blocking entry into the peripubertal period, still are not known. The present study was conducted to investigate possible neurotoxic effects of prepubertal ETOH exposure on specific hormones which are known to be extensively involved in the early pubertal process. Therefore, we have examined the chronic effects of ETOH in immature female rats just prior to entering the peripubertal period of development,
Follicle stimulating hormone
on the hypothalamic contents of growth hormone releasing hormone (GHRH), somatostatin (SRIF) and luteinizing hormone releasing hormone (LHRH), as well as the serum levels of growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH). METHOD
Animals and Surgery Immature female Sprague-Dawley rats of the Harlan line (Harlan, Inc., Houston, TX) were used. Each rat was anesthetized at 23 days of age with Equithesin (Fort Dodge Labs. Inc., Fort Dodge, IA) and surgically implanted with a permanent intragastric cannula by a procedure that we have described previously (11). The animals were then housed individually under controlled conditions of light (lights on, 0800 hr; lights off, 1800 hr) and temperature (23°C), with ad lib access to food (Purina Laboratory Chow) and tap water. Animals were allowed to recover from surgery for five days prior to the beginning of the experiment. In
~Requests for reprints should be addressed to W. Les Dees, Laboratory for Neuroendocrinology, Department of Anatomy, College of Veterinary Medicine, Texas A&M University, College Station, TX 77834.
21
DEES ET AL.
22
order to assess the effects of ETOH on specific hormones known to be involved in the early pubertal process, each rat was administered a specific diet regimen, then killed in the anestrous phase of puberty as described below.
Experimental Procedure Twenty-eight-day-old rats were weighed and assigned to one of three groups, each averaging between 60~65 g body weight. Group one consisted of 30 animals that received a 5% ETOH liquid-diet and group two consisted of 14 animals that received the isocaloric control liquid-diet (Bio-Serve, Inc., Frenchtown, NJ). Group three served as an additional set of controls which consisted of 15 animals that were cannulated and maintained on Lab Chow and water, ad lib, throughout the experiment. On day 29, the animals in groups I and II began receiving their respective liquid-diet regimen. These diets were administered in such a manner that 7.5 ml of the respective diet was injected via the intragastric cannula (5 injections of 1.5 ml each) equally dispersed over the lights-on period, then no more than 30 ml of diet was available ad lib (bottle) during the lights-off period. In order to provide adequate food supply for these immature growing animals, beginning on day 30 and again on days 32 and 34, the amount of diet injected via the cannula was increased by 0.5 ml per injection. Also, at these same times the amount of diet made available each night in the bottle was increased to 35, 40 and 50 ml, respectively. Thus, by day 34, each animal was receiving the maximum of 5 intragastric injections (3.0 ml each) of diet during the lights-on period and 50 ml of diet, ad lib, during the lights-off period. We have reported previously that this procedure enabled the liquid-diet control group to grow at exactly the same rate as the Lab Chow control group (7,10). Additionally, this method not only ensured that all liquid-diet fed animals received approximately the same number of calories per day, but also ensured that all of the ETOH-treated animals received the same amount of ETOH per day. Both the control and ETOH-treated animals were weighed and killed by decapitation between 0800 and 0900 hr at a mean of 34.0_+ 1.1 and 34.5_+ 1.3 days of age, respectively. The uterus and ovaries from each animal were removed and weighed to the nearest milligram and all animals were confirmed as being in the anestrous phase of puberty based on well-defined criteria which classify the different phases of the peripubertal period (1,15). Briefly, animals in the anestrous phase had closed vaginae, small uteri (up to 94 rag) and no intraluminal fluid. Trunk blood was collected, centrifuged at 3000 × g for 20 min, and the serum stored at - 20°C until assayed for GH, LH, and FSH. Brains were removed and hypothalami (with median eminence) excised by dissection using the pyriform lobes, caudal borders of the optic chiasm and mammillary bodies, and the thalamic adhesion as the lateral, rostral, caudal, and dorsal boundaries, respectively. The brain tissues were immediately frozen on dry ice and stored at - 7 0 ° C until processed further for assaying the hypothalamic contents of GHRH, SRIF and LHRH.
14ypothalamic Extracts and Peptide R1A's Each hypothalamus was lyophilized and subjected to acetic acid extraction for peptides by a procedure previously described (14). After the addition of 500 tzl of 2 N acetic acid, each sample was vortexed for one min and placed in a boiling water bath for five min to destroy protease. The homogenates were centrifuged at 4 5 0 0 × g for 30 min, and 300 ixl of supernatant fluid was removed and frozen. Samples were similarly extracted two more times (without boiling) with 500 Izl 0.1 N acetic acid, and 500 and
400 ixl, respectively, of supernatant fluid was removed after each centrifugation. The total supernatant fluid for each sample was lyophilized and stored at 4°C under desiccant. Each extract was reconstituted just before assay with 1 ml of phosphate-buffered saline pH 7.4. Extracts were measured for their GHRH content using a specific RIA. The antiserum used (RAS 8068) was raised against synthetic rat GHRH (rGHRH, Peninsula Labs, Belmont, CA) and does not cross-react (
