Growth patterns and hormonal profile of male rats with protein-calorie malnutrition.код для вставкиСкачать
THE ANATOMICAL RECORD 197:339-354 (1980) Growth Patterns and Hormonal Profile of Male Rats With Protein-Calorie Malnutrition DAMON C. HERBERT Department of Anatomy, The University of Texas, Health Science Center at San Antonio, San Antonw, Texas 78284 ABSTRACT A condition of protein-calorie malnutrition was precipitated in young Sprague-Dawley male rats at 20 days of age using an 8% low protein diet (LPD). At five-day intervals for up to 50 days of age, the rats were studied to determine the effect of a n LPD on the reproductive axis of the endocrine system. Daily monitoring of the body weight, as well a s the consumption of food, kilocalories, and protein was conducted. The same parameters were followed over the identical time period in a group of animals designated as controls which were fed a standard laboratory diet (SLD) containing 27% protein. The controls showed a linear growth rate over the 30-day experimental period. In comparison, the malnourished rats grew more slowly so that by 50 days of age, their mean body weight was 68.9 5 3.1 g as compared to 249.1 t 6.1 g for the controls. The daily food, kilocalorie, and protein intake by the experimental animals were also appreciably less. The pituitary gland, ventral prostate gland, testes and liver were smaller in the animals fed the LPD. This was observed as early as five days after initiating the dietary regimes and remained a consistent observation until the end of the experiment. In general, the absolute weights of these organs in the 50 day-old malnourished rats were similar to those found in 25 to 26 day-old animals fed the SLD. The relative weights of the pituitary gland and liver remained similar between the two animal groups. The testes and ventral prostate gland, however, were relatively smaller in the malnourished animals a t nearly every time interval studied. On light microscopic examination of the testes, it was found that normal maturation of the germ cells failed to occur in all but one of the experimental animals, whereas maturation proceeded normally in the rats fed the SLD. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), and testosterone were lower in the malnourished animals at all ages studied. These hormones did not exhibit the fluctuations that were seen in the controls and are typical in rats that are becoming sexually mature. The effect of protein deficiency on the concentration of the pituitary gonadotrophins was more varied. FSH concentrations were consistently lower, PRL was moderately affected, and LH remained essentially unchanged. Hypothalamic LH-releasing hormone was measured and found to be significantly less in the rats fed the LPD a t most of the time intervals examined. axis is impaired These results indicate that the hypothalamo-hypophyseal-gonadal when the consumption of proteins and calories is decreased. The possible involvement of extrahypothalamic centers in the control of hormone secretion in the protein-deficient rat is discussed. A large number of reports have been published describing the complications and deleterious effects of protein-calorie malnutrition (PCM) in man. It is well established that this problem abounds in countries that are overpopulated. Despite the apparent paradox, reproductive function is impaired in individuals with PCM (Brasel, '78). 0003-276x/80/1973-0339$02.90 0 1980 ALAN R. LISS. INC. The relationships between reproduction and nutrition have been studied in laboratory animals by employing diets deficient in or totally devoid of protein, or under conditions where animal subjects have been chronically underfed (semistarved). Early reports from the work Received May 23, 1979; accepted October 8, 1979. 339 340 DAMON C. HERBERT of Evans and Bishop ('22) established the need for adequate dietary protein in order to initiate and maintain regular estrous cycles in the female rat. In the male, puberty was found to be delayed when animals were given a proteinfree diet (Horn, '55). If, however, they were refed a diet containing either 6%or 1810 casein for 30 days, 25% and Two, respectively, of the animals became sexually mature. The absolute weights of the gonads and accessory reproductive organs were consistently lower in male rats a) given a protein-free diet (Horn, '55; Srebnik and Nelson, '62; Srebnik, '641, b) starved for seven days (Mulinos and Pomerantz, '411, or c) chronically underfed (Mulinos and Pomerantz, '40, '41; Grewal e t al., '71; Howland, '75). The most severe changes were noted in the weights of the accessory organs (Mulinosand Pomerantz, '41; Srebnik and Nelson, '62; Srebnik, '64). Moreover, it was reported by Srebnik and Nelson ('62) that the debilitating effects of a protein-free diet were more pronounced i n younger than in older animals. Pomerantz and Mulinos ('39), (Mulinos and Pomerantz, '40) termed the malnourished condition associated with chronic underfeeding "pseudo-hypophysectomy." Atrophy of the reproductive organs was quite pronounced in their animals but to a lesser degree than that observed in hypophysectomized rats. This observation was later substantiated by Srebnik ('64) who found larger testes, seminal vesicles, and prostates in males fed a protein-free diet as compared to hypophysectomized rats. These findings indicated that the anterior pituitary gland was exerting a trophic effect on the reproductive tract, even in the absence of dietary protein. Factors controlling the secretion of the anterior pituitary gonadotrophins and the gonadal hormones have been shown to be sensitive to dietary manipulations. The pituitary content of luteinizing hormone (LH) and folliclestimulating hormone (FSH) was reduced a s a result of protein deprivation (Srebnik and Nelson, '62), but was more variably affected by chronic underfeeding or starvation (Meites and Reed, '49; Root and RUSS,'72; Howland and Skinner, '73; Howland, '75; Nakanishi et al., '76). Feed restriction for periods of 30 or 60 days has been reported to decrease pituitary prolactin (PRL) levels (Nakanishi et al., '76). All three gonadotrophins were lower in the serum of rats either starved for seven days (Howland and Skinner, '73; Campbell et al., '77) or underfed for varying Deriods of time (Howland, '75; Stewart et al.. '75; Campbell e t al.,'77). Similarly, these two forms of malnutrition also had an adverse effect on testosterone secretion (Grewal et al., '71; Howland, '75). The most prevalent forms of malnutrition in today's world are thought to be related to a deficiency of protein in the diet and not due to starvation or consumption of a protein-free diet. In addition, the largest single group of undernourished persons are children (WHO, '72). In view of this, it was of interest to examine the effect of a low protein diet on the development of the reproductive system in animals that were maturing sexually in a n effort to gain information that could be related to preadolescent children suffering from PCM. Such studies have been conducted using female rats (Evans and Bishop, '22) but, heretofore, have not been thoroughly examined in the male. The present report details changes that have occurred in the hypothalamo-hypophyseal-gonadalaxis of protein-calorie malnourished male rats a t fiveday intervals between 20 and 50 days of age. MATERIALS AND METHODS Twenty day-old Sprague-Dawley male rats were purchased from Simonsen Laboratories, Inc., Gilroy, CA. They were housed four animals per cage in air-conditioned quarters with a light-dark cycle of 14:10, controlled temperature of 22-24"C, and humidity regulated a t 500/0.Food and water were provided ad libitum'. The animals were separated into 13 experimental groups with each group containing eight animals. Animals i n the first seven groups served a s controls and were fed a standard laboratory diet (SLD) while rats in the remaining six groups were given a low protein diet (LPD). Both diets were purchased in pellet form from ICN Pharmaceuticals, Inc., Cleveland, OH. The SLD consisted of 27%protein in the form of vitamin free casein, 5w0 starch, 10% vegetable oil, and 4% salt mixture USP XIV. The experimental LPD was composed of 8% protein (vitamin free casein), 7810 starch, lWo vegetable oil, and 4% salt mixture USP XIV. The SLD was provided with 4.3 Kcal per gram and the LPD 4.5 Kcal per gram. A special vitamin fortification mixture, prepared by ICN 'Since the main objective ofthe study was to induce protein-ealorie malnutrition and nottostudytheefleetsofdeficiency ineitherproteins or calories alone, the rats were not pair-fed. Moreover, when pairfeeding is employed. the control animals are fed less than their normal amount of food and are, therefore, partially protein and/or calorie malnourished,aemistarved, and ntrensed. Both stress (Eukereta].. 75) and semistarvation (Campbell et el.. 7 7 ) have been reported to atTed pituitary hormone secretion 341 PROTEIN-CALORIE MALNUTRITION IN MALE RATS Pharmaceuticals, Inc., was incorporated into each diet in an amount of 1 kg per 45.5 kg of diet. The changes in body weight and the amount of food consumed by each rat were monitored on a daily basis. The latter calculation was made by weighing the amount of food present each morning, subtracting this from the amount of food measured at the same time on the previous day, and dividing by eight. Additional computations were made from these data to determine the daily intake of protein and kilocalories (Kcal)by each rat. These data were statistically analyzed by analysis of variance and by the “t” test for comparison of differences between means. One group of control animals was sacrificed immediately upon arrival. The remaining control and the experimental rats were sacrificed a t five-day intervals beginning a t 25 days of age and continuing through 50 days of age. At each of these intervals, eight control and eight experimental animals were sacrificed. This provided exposure of the rats to either the SLD or the LPD for periods of 0 to 30 days over the ages of 20 to 50 days. All animals were sacrificed by decapitation between 0800 and 1100 hours. Trunk blood was collected, allowed to clot, and the serum collected and stored a t -20°C. The following organs were then removed from each animal and weighed: the hypothalamus, pituitary gland, testes, ventral prostate gland, and the liver. The testes were fixed for 72 hours in Bouin-Hollande solution, embedded in paraffin wax, and stained with hematoxylin and periodic acid-Schiffs reagent. The hypothalamus was placed in 1m12 N acetic acid and homogenized. This was subsequently heated in boiling water for five minutes, centrifuged, and the supernatant collected for lyophilization. The pituitary gland from each rat was hemisected along the midline. Half was homogenized in a 0.01 M phosphate-saline buffer, pH 7.0, and the other half fixed for histologic examination (the details of these findings will be the subject of a future report). The pituitary homogenates along with a portion of the sera samples were assayed for LH, FSH, and PRL, using double antibody radioimmunoassays. The LH assay procedure was similar to the one described by Niswender et al. (’68),while the FSH and PRL assays were carried out using reagents supplied by the NIAMDD. The lyophilized hypothalami were reconstituted in 1ml distilled water, and the content of LH-releasing hormone (LHRH) measured using a modification of the procedures described by Arimura et al. (’73)and Nett and Adams (’77).The LHRH antibody was generously supplied by Dr. Terry Nett. Serum total testosterone were quantified using a modification of the assay procedure employed by Pauerstein et al. (’78).The antibody to testosterone was purchased from Endocrine Sciences. The coefficient of variation in the testosterone assay was 2.0%. All assay and organ weight data were pooled according to experimental grouping and statistically evaluated as previously described. RESULTS Body growth and development The initial body weight of the male rats a t 20 days of age ranged from 44.5 to 46.6 g, with a n average of 45.3 ? 0.2 g. No statistical differences were present among the 13 animal groups. The changes in the body weight of the control rats fed the 27% protein diet were linear over nearly the entire 30-day experimental period (Fig. 1).In contrast, the animals given the LPD gained weight a t a much slower rate, which was neither linear nor parallel to that of the controls. These animals had an irregular growth pattern, gaining a small amount of weight over a few days, followed by a brief 250 - 200 - -E 2 150- Em .- 2” s” loo- m 0 J , , , , , , , x) 25 30 35 40 Age(doys) 45 50 Fig. 1. Mean body weight (-t SE) of the control (solid circles) and experimental (open circles) rats at fiveday time intervals between 20 and 50 days of age. 342 DAMON C. HERBERT Fig. 2. A control and an experimental rat at 50 days of age. Patchy hair loss can be noted on the neck of the malnourished rat. PROTEIN-CALORIE MALNUTRITION IN MALE RATS w 701 60 - I v 50- -5 40- -4 0 3 8 Y u) 3020 - -3 a r - '"1, * p- 0 20 ? 0, 8. , 25 -2 - -=: - *--*--P--c---a x) , 35 , 40 , 45 , 50 Age (days) Fig. 3. Mean ( 2 SE) intake of kilocalories (solidline) and grams protein (broken line) of animals fed either the SLD (solid circles) or the LPD (open circles). period of no weight gain, and then another episode in which there was a n increase in body weight. At the end of the experimental period, the average body weight of the animals fed LPD was 68.9 +- 3.1 g which was similar to the weight of 24 day-old control rats (67.5 +- 1.4 g). Figure 2 illustrates a control and an experimental animal which had been fed their respective diets for 30 days. A small amount of hair loss is apparent on the dorsal and lateral sides of the neck of the malnourished rat. This was noted in 23 of 48 animals fed the LPD. In general, it was confined to the dorsal or the ventral aspects of the trunk of the animal and was typically seen in patches. No external parasites could be found on these animals that would have resulted in the alopecia. Thinning of the hair has previously been reported in animals fed an LPD (Tulp et al., '79). No hair loss was observed in the controls. Food, kilocalorie, and protein consumption The Kcal and grams protein consumed by each rat in its respective experimental group are shown in Figure 3. Curves representing the daily food intake are not illustrated, but were found to nearly parallel those depicting the Kcal consumed by both the control and the malnourished rats. The animals fed the SLD consumed food, and therefore had an intake of Kcal and proteins, at a rate that was distinctly different from that observed in the experimental rats. These parameters were linear in the controls for up to ten days (30 days of age) after which the rate of food consumed and intake of Kcal and grams protein began to de- 343 crease. Despite this, the weight of the rats continued to rise linearly (Fig. 1). In the rats fed the LPD, there was a n initial decline in the amount of food, Kcal, and grams protein consumed which was then followed by a 10-day period in which there was no change. After 35 days of age, however, a linear increase in the daily intake of food, Kcal, and protein was noted. At all time intervals examined, these parameters were consistently lower in the rats fed the LPD as compared to the controls. At the end of the experiment, the food and Kcal consumed by the malnourished rats was approximately the same as that observed in 22 day-old rats maintained on the SLD. Absolute and relatiue organ weights Table 1 contains the absolute weights of the pituitary gland, testes, ventral prostate gland, and liver in both the control and experimental animals. All four organs increased consistently in weight over the 30-day period in the control animals. In most instances, the increases were significantly greater a t each successive time interval. The major exception to this was in the pituitary gland weight where the changes, while greater at each five-day time interval, were statistically significant only when comparing the values measured on day 25 with day 30, or day 30 with day 35. In the malnourished animals, consumption of the LPD led to more erratic changes in the pituitary gland, testes, ventral prostate, and liver during the 30-day experimental period. Moreover, the weights of these organs were always significantly less (P < 0.001) than those recorded for the controls a t all of the time intervals examined. A significant reduction (P < 0.02) in pituitary gland weight occurred in the experimental animals between 20 and 40 days of age. Over the remaining 10 days of the study, a slight increase was noted, so that by 50 days of age, the pituitary gland had obtained a weight which was similar to that found in the 25 day-old control rats. There was no significant change in testicular weight of the malnourished animals for the first 20 days of the study. After this period of time, a marked increase was recorded; however, the growth rate was less than that observed in the controls. At the end of the experiment, the testes were similar in weight to those of a 26 day-old rat maintained on the SLD. The absolute weight of the ventral prostate gland in all of the experimental animals was consistently less than the value recorded in the 20 day-old rats. Between 20 and 30 days of age, there was a significant decrease (P < 0.005) in prostatic weight, which was then followed by a 344 DAMON C. HERBERT TABLE 1. Absolute organ weights in protein-calorie malnourished male rats Pituitary gland (mg) Age (days) 20 C M C 2.3 t 0.2" - 303 t 16 25 2.5 t 0.2 30 35 5.6 0.2 540 265 48.1 28.9 i24' t 25 t 2.5' t 1.7 1.6 0.1 855 f42 t 31 ? 1.6 297 66.5 5.27 t 0.9 2 94.4 7.9" t 1.9 137.9 i 9.5' f 261 t 28 1.6 0.1 1648 t4w t28e 6.8 1.9 201.5 t 0.2 2089 t 33' 457 t 0.5 t 486 t- 16.01 2256 556 611 47d 260.0 21.1 i- i 13.6 t 2.8 i- 7.1 t 0.4 5 2.5 0.26 354 * M - C 2.5 i 0.1 3.5 2.0 t 0.1h 21.1 2 1235 6.1 Liver (PI M - C 31.3 t 1.6 t 4of i 0.3 50 M - * 0.1 ? 45 1.7 Ventral prostate gland (mg) * 0.1 3.7 t 0.2 40 Testes (mg) f 0.1 6.0 * 0.1' 23.2 k 7.1 _~0.3~ t 0.1 10.3 0.4' t 0.1h 20.1 2.6 2 23.9 3.3 t 0.4b 2.5 2.9 11.3 2 2.3 0.1 2 12.0 2.7 0.2 3.3 * 0.4 f 0.2e C = control rats fed the SLD M = malnourished rats fed the LPD =mean 2 SE P .C 0.05 (statistical comparisons were made between the mean for each organ welght and the weight of t h a t organ at the previous time Interval) 'P < 0.02 "P < 0.01 *P < 0.005 ' P < 0.001 TABLE 2. Relative organ weights in protein-calorie malnourished male rats Pituitary gland (mg) C M C M 4.6 - 624 t 34 - i 0.4' 25 30 3.4 3.6 757 512 67.3 f 0.2 t 29 t 56'' t 2.4 788 43 596 61.1 t63 t 4.9 876 38 510 66.9 i 53g t 5.4 866 29 t 629 49 t 6.6 739 144d t 7.9 2 822 45 ? 104.6 5.6 t 4.F 3.4 50 = M = t 2.9 0.2 ? 2 k 3.2 948 f 0.4 t 30 2.4 f 0.2 C 3.2 3.3 * 0.3 3.1 0.3 t 0.1h 3.2 t 0.1 45 5 3.9 i 0.2 40 M - C 63.5 t 3.8 -c 0.3 t 0.1 35 Ventral prostate gland (mg) Testes (mg) 5 3.6 0.3" 907 21 k t t 72.8 95.9 62.7 3.7 2 Liver (g) C M 5.0 t 0.2 - 2 42.8 4.9 0.1 5.6 4.5 f 0.2 4.7 * 2.P t 0.1 45.3 2.7' 5.0 4.9 i:0.1 t 0.1 t 35.9 t 0.2': * 4.8 5.4 5.2 t 0.2 2 0.2 37.2 4.3 t 0.1 5.3 30.7 t 4.8 0.1 4.4 t 0.18 4.8 t 0.2 control rats fed t h e SLD malnourished rats fed the LPD dmean i SE. Organ weights are expressed per 100 g body weight. DP< 0.05 (statistical comparisons were made between means from control and experimental animals at each time interval for each organ) P < 0.025 dP < 0.02 e P c 0.01 I P < 0.005 'P c 0.001 PROTEIN-CALORIE MALNUTRITION IN MALE RATS period in which there was no additional change. An appreciable reduction (P < 0.005) in liver weight also occurred over the first five days in the animals fed the LPD. However, between 25 and 50 days of age, the liver enlarged and reached a weight which was very similar to that observed in the 25 day-old control rats. The relative organ weights are summarized in Table 2. The weight of the pituitary gland gradually decreased in the controls as they aged. A similar observation was made in the malnourished rats between 20 and 40 days of age. For the remaining period of the study, an increase in pituitary weight was recorded. In spite of this, the pituitary gland of the 50 dayold malnourished r a t s was significantly smaller (P < 0.02) than that found in the controls a t the onset of the study. When comparing the values for pituitary gland weights in the controls with the experimental animals at each of the ages studied, a significant difference was found only at 35 and 50 days of age. At a majority of the time intervals sampled, the relative weights of the testes and ventral prostate glands were statistically greater in the rats maintained in the SLD. Moreover, these two organs continued to enlarge relative to the body weight in the controls over the 30-day experimental period. There were no significant changes in testicular weights in the protein deficient animals until after 40 days of age. This is in marked contrast t o the decrease recorded for the weight of the ventral prostate gland which occurred throughout the course of the study. There were fluctuations in liver weight in both the control and experimental animals. Significant differences between the two animal groups were found at only two time intervals. At 50 days of age, the weight of the liver was essentially the same in both animal groups, and was not significantly different from the weight recorded in the control rats a t the beginning of the experiment. Histology of the testes Histologic examination of the testes revealed that the diameter of the seminiferous tubules was less, and the size and number of Leydig cells were decreased in the animals fed the LPD (Figs. 6-11). In addition, a disruption of the overall integrity of the tubular epithelium was evident. These changes were first observed as early as five days following the onset of the study (Figs. 6,7),and continued to be prevalent as long as the LPD was administered. Spermatids in the Golgi phase could first be differentiated in the controls at 25 days of age (Fig. 6). Five days later, more mature spermatids 345 were easily distinguished in all of the animals maintained on the SLD. Spermiogenesis progressed normally in the controls (Figs. 8, lo), with immature spermatozoa (stage 19 spermatids) being observed in the seminiferous tubules a t 45 and 50 days of age (Fig. 10). All but one of the animals maintained on the LPD did not undergo puberty. Golgi phase spermatids predominated in the testes of the experimental animals from 2 5 4 0 days (Figs. 7,9).At 45 days of age, after being on the LPD for 25 days, acrosomal phase spermatids were present in one animal, mature phase in another, and either cap or Golgi phase spermatids in the remaining six. By 50 days of age, five of the eight rats had mature phase spermatids in their seminiferous tubules (Fig. 11).A large number of stage 19 spermatids were observed in only one of the five, indicating that it was sexually mature. The remaining 50 day-old animals displayed more immature forms of the spermatids in their seminiferous tubules. Serum gonadotrophins The concentration of circulating LH, FSH, and PRL are shown in Figure 4. LH increased by 107% in the control animals during the first five days of the study and rose again by 116% between 35 and 40 days of age. No significant changes were found from days 25 to 35 or after 40 days of age. Fluctuations in serum LH occurred in the malnourished rats throughout the entire experimental period; however, no two values were found to be statistically different. LH in these animals was consistently lower than those values recorded for the rats fed the LPD. Serum concentrations of FSH increased from 370 20 to 526 2 47 ngiml in the control animals during the five day period between 25 and 30 days of age. No additional vacillations occurred until after day 40. Thereafter, a significant decrease was recorded. In contrast, FSH declined very rapidly in the protein deficient animals from the onset of the study until day 25. The hormone levels then plateaued and remained essentially unchanged for the duration of the experiment. By 50 days of age, FSH in the animals was 5wo less than the value measured in the age-matched controls. PRL rose throughout nearly the entire 30day experimental period in the rats maintained in the SLD. Likewise, an increase in hormone levels occurred between 20 and 40 days of age in the malnourished rats. However, the rate of changes in serum PRL in these animals was significantly less than that which transpired in the control animals. * 346 DAMON C. HERBERT 20 25 30 35 45 40 50 T 0 ' $0 215 40 I I T I 35 40 45 50 Age (days) Fig. 4. Serum concentration of LH, FSH, and PRL in control (solid circles) and malnourished (open circles) rats from 20 to 50 days of age. Each point representsthe mean ? SE. Statistical comparisons were made between means from control and malnourished animals at each time interval. Pituitary gonadotrophins The content of the pituitary gonadotrophins was, with one exception (LH on day 251, statistically greater in the control animals a t each time interval sampled. The mean hormone values in the two animal groups on day 50 were: LH, 79.1 2 14.5 vs. 12.2 5 1.0 pg (controls vs. experimentals); FSH, 55.5 t 5.3 vs. 11.4 t 2.1 pg; PRL, 610.1 t 84.9 vs. 97.4 21.6 ng. * Table 3 summarizes the concentrations of pituitary LH, FSH and PRL. There was approximately a two and one half fold increase in LH levels in the pituitary glands of the rats fed the protein-enriched SLD from the beginning of the experiment until day 35. Five days later, a sharp decline was detected which coincided with a marked increase in circulating LH (Fig. 4). At the last two sampling times, LH values were similar to those recorded on day 35. In the experimental rats, pituitary LH concentrations displayed minimal variation from one five-day interval to another. The one age at which there was a deviation from this was on day 45 when hormone values increased by 88%.In comparison to the control rats, pituitary LH differed significantly only at 35 and 50 days of age. The concentration of pituitary FSH in the rats fed the SLD followed a similar pattern as that described for serum FSH. Higher values were present in the middle of the experiment than a t either the onset or a t the termination of the study. Aside from day 40 (P < 0.021, FSH remained essentially unchanged in the malnourished rats. However, the FSH values in these animals were consistently and, with one exception, significantly different from those measured in the controls. During the first half of the experiment, PRL rose by 338% in the pituitary glands of the control animals. Thereafter, hormone levels vacillated considerably. By day 50, they had reached their peak value of 105.3 ? 18.7ng/mg. Significant changes in pituitary PRL were not measured in the protein deficient experimental animals until after 40 days of age, at which time a two-fold increase was observed. At three of the six intervals studied between days 25 and 50, PRL concentrations were statistically different from the control values. Serum testosterone The levels of total testosterone in the sera of the control and malnourished rats a r e presented in Table 4. Hormone concentrations were similar in the control animals until after 40 days of age. The values recorded on days 45 and 50 differed significantly from those measured a t 40 (P < 0.025) and 45 (P < 0.001)days of age, respectively. Total testosterone was detectable only on days 25,45, and 50 in the sera of the protein deficient rats. At the other ages sampled, hormone levels were below the minimum level of sensitivity for the radioimmunoassay. No statistical differences were observed between the two animal groups that were 25 days old, but the values measured on days 45 and 50 were significantly greater in the rats given the SLD. 347 PROTEIN-CALORIE MALNUTRITION IN MALE RATS Hypothalamic LH-releasing hormone There was a general increase in the content of LHRH in the control hypothalami as the animals aged (Fig. 5 ) . In comparison, sharp fluctuations were recorded in hypothalamic LHRH in the malnourished rats; the only highly significant change (P < 0.001) was a 71% rise between 45 and 50 days. A greater amount of LHRH was consistently found after day 25 in all animals fed the SLD. When these results are expressed in terms of hormone concentration (Pg LHRH/mg hypothalamus), a significant difference between the two animal groups was detected from 30 to 45 days of age (Fig. 5 ) . DISCUSSION In the present report, the reproductive axis of the endocrine system was studied in malnourished male rats over a period in which marked hormonal changes have been shown to occur (Negro-Vilar et al., '73; Dohler and Wuttke, '75) and when the onset of puberty was expected. The observations represent the first comprehensive analysis of the interrelationships between hormone secretion and nutrition in sexually maturing animals. Previous investigators have focused their endocrine nutrition studies almost entirely on the adult r a t (Mulinos and Pomerantz, '40, '41; Srebnik and Nelson, '62; Srebnik, '64, '70; Root and Russ, '72; Howland and Skinner, '73; Howland, '75; Campbell et al., '77). The diet that was fed our experimental animals contained a similar amount of energy (4.5 Kcal per gram) as that present in the control SLD (4.3 Kcal per gram), but provided 70.4% less protein. Due to the differences in the amounts of food consumed by the two groups of rats, the malnourished animals also became deficient in their daily intake of calories within a very short period of time. The resultant effect was a malnourished state precipitated by insufficient calorie and protein consumption. This led to a dramatic decrease in the growth rate and pattern of the developing animals. By 50 days of age and after having been fed the LPD for 30 days, the weight of the malnourished rats was less than one third that of the controls. In a similar study, Tulp and coworkers ('79) recorded a 730decrease in body weight in male Fig. 5. Mean (t SE) LHRH content (top) and concentration (bottom) of hypothalami from 20 to 50 day old control (solid circles) and malnourished (open circles) rats.StatisticaI comparisons were made as described in the legend for Figure 4. I I I I I I I 20 25 30 35 40 45 50 Age (days) 348 DAMON C. HERBERT TABLE 3. Concentration of the pituitary gonodotrophins LH M C 5.3 1.5" 4.7 f 0.6 30 f 2 7.5 1.0 5.7 12.0 4.3 43.6 f 8.0 t 0.5' i 2.8 2 12.3 ? 4.1 0.7' t 1.0 ? 5.9 0.4 ? 9.5 0.8 ? 2.4 0.7" 12.7 2.0 ? 11.1 1.5 ? 8.1 1.0 6.1 r 1.3 11.1 1.8 ? 5.1 0.5" ? 7.7 0.5 f 0.6" 7.6 t 11.2 4.3 21.7 36.1 9.3 i 5.9 ? f 0.3 t 0.8 40 50 3.7 i 0.5e t 1.7 5.9 0.5d ? 45 8.4 5.3 0.4 M C 16.0 ? 3.4t' ? 10.7 0.9 35 M C 4.1 t 0.5" 25 PRL FSH 20.3 4.7e ? 70.2 16.1 3.4e ? 23.4 27.4 3.7 t 3.2 2 61.7 49.9 2 8.5 t 7.8 105.3 45.9 5 18.7 f 9.6' C = control rats fed the SLD M = malnourished rats fed the LPD 'mean 2 SE, pgimg %ghg P < 0.02 (statistical comparisons were made between means from control and experimental animals at each time interval for each hormone) dP < 0.005 ( P < 0.001 (. TABLE 4 . Total serum testosterone Age Control rats Malnourished rats 20 0.74 t 0.08" - 25 30 35 40 0.31 0.36 0.38 f 0.03 i 0.03 t 0.04 ? 0.25 i 0.20 < 0.20 < 0.20 i 0.01 0.79 0.10 45 50 1.65 * 0.30 0.27 f 0.01b 2 3.42 0.77 0.27 2 0.02" "mean 2 SE, nglml bP< 0.001. Statistical comuarisons were made as described in Table 3. rats fed a n 8%protein diet from 21 to 53 days of age. Widdowson and McCance ('63) studied the growth patterns of male rats that were underfed between three and six weeks of age. At the end of their experimental period, they reported that the malnourished rats were ". . . over 60 grams behind the well nourished ones and were less than half their weight." In spite of their reduced food intake, these animals grew at what appeared to be a steady and rather constant rate over the three-week period (Widdowson and McCance, '63). This was not true for rats fed a n LPD (Fig. 1;Tulp et al., '79).For the first 15 days of our study, the total increase in body weight for each rat was 6.2 grams, while over the latter phase of the experiment, each Figs. 6, 7. Sections of testes from a control (Fig. 6) and an experimental rat (Fig. 7) at 25 days of age. The number of developing germ cells is less in the seminiferoustubules of the malnourished rat. In addition, many of these cells have become detached from the Sertoli cells and appear "freefloating" individually or in clusters within the lumen of the tubules. x 200. (Insert x 375.) PROTEIN-CALORIE MALNUTRITION IN MALE RATS 349 350 DAMON C. HERBERT Fig. 8, 9. Sections of testes from a control (Fig. 8)and an experimental rat (Fig. 9) at 40 days of age. Maturation phase spermatids are present in the control testis (asterisk)but are absent in the seminiferous tubules of the rats fed the LPD. In these animals, the most mature spermatids were in the Golgi phase. x 200. (Insert x 300.) PROTEIN-CALORIEMALNUTRITION IN W E RATS 351 Fig. 10, 11. Sections of the testes from a control (Fig. 10) and an experimental rat (Fig. 11) at 50 days of age. Step 19 spermatids are only present in the seminiferous tubules of the control animal. X 200. (Insert x 300.) 352 DAMON C. HERBERT rat gained 17.7 grams. These changes can be attributed to the differences in eating habits in each of the two halves of the 30-day study. A significant increase was not recorded in the amount of food, proteins, or calories consumed between 20 and 35 days of age. In contrast, a rather marked rise in all three parameters was noted between 35 and 50 days of age. The factors controlling these changes are not fully understood. The differences between the two animal groups in the absolute weights of the pituitary gland, gonads, ventral prostate gland, and the liver were striking. All four organs were consistently smaller in the experimental rats. One intriguing aspect was the rapid onset of the malnourished state which was accompanied by an initial decrease in all of the organ weights recorded. With the exception of the ventral prostate gland, there was a recovery from the initial weight loss. The time span preceding the onset of the recovery was the shortest in regard to the liver weight and the longest for the pituitary gland. At the end of the study, the weight of the pituitary gland, gonads, and liver was similar to that of normal rats that were 26 days of age or younger. The ventral prostate gland was severely atrophic and weighed 32.6% less than that measured for the 20 day-old controls. The effect of protein deficiency on the hypothalamo-hypophyseal-gonadalaxis cannot be ascribed solely to inadequate dietary proteins and calories. The relative liver weight, which was used as a n index to determine the general systemic effects of the two diets, was similar in both animal groups throughout a majority of the study. Rather, the data collected from the experimental animals suggest the existence of multiple hormonal deficiencies which commenced almost immediately after the introduction of the LPD and were sustained as long as the malnourished condition existed. The patterns of gonadotrophin secretion during the 30-day period between 20 and 50 days of age were markedly different in the protein-calorie malnourished rats as compared to normal animals of the same age. There was a n elevation in LH, FSH, and PRL in the serum of the rats fed the SLD between 20 and 40 days of age; thereafter, either no major changes transpired or, as was the case with FSH, hormone levels decreased. Similar fluctuations in peripheral gonadotrophins have been reported in the male rat by Dohler and Wuttke ('75). Malnutrition, on the other hand, caused a fall in serum FSH, very pronounced vacillations in LH levels and only a moderate increase in peripheral PRL concentrations. By 50 days of age, only PRL was found to be elevated above values measured a t the onset of the study. LH was 39% lower and FSH had decreased by 5wo. As a consequence, the circulating levels of the gonadotrophins were too low to initiate and maintain normal growth and development of the gonads in the malnourished rats. The relative testicular weights, the diameter of the seminiferous tubules, and the size of the interstitial cells of Leydig were all smaller. The physiologic activity of these cells was severely depressed which was reflected not only by lowered serum testosterone values but also by a marked atrophy of the ventral prostate gland. Ultimately, a condition of hypopituitarism evolved which resulted in a failure of these animals to become sexually mature. Other investigators have found one or more of the gonadotrophins, as well as testosterone, to be affected by starvation (Grewal et al., '71; Howland and Skinner, '73; Campbell et al., '771, chronic underfeeding (Howland, '75; Stewart et al., '75; Campbell et al., '771, or protein deprivation (Srebnik, '70). Glass et al. ('79) have recently reported that a 90protein-containing diet fed to rats from 21 to 50 days of age failed to significantly affect either serum LH or testosterone, but did reduce circulating FSH levels. They were able to measure a decrease in the release of testosterone by gonads from malnourished rats that were cultured either alone or in the presence of human chorionic gonadotrophin. The morphology of the seminiferous tubules was normal in their animals, which is in contrast t o our findings and those of Horn ('55). Glass and colleagues ('79) did note a n effect of their diet on sex accessory organ weights. The reason(s) for the discrepancies between the present report and that of Glass et al. ('79) is (are)unclear. It seems unlikely that one can account for these differences solely on the basis of the strain of animal studied [SpragueDawley (ours) vs. Wistar (Glass et al., '7911. Another possible explanation may reside in the percentage of protein within the two diets. The additional 1% they employed may have exceeded a minimal threshold of dietary protein needed to sustain "normal" reproductive function and to stimulate puberty to occur, even though FSH levels were reduced and sex accessory organ weights were well below those found in the controls. The events controlling hormone synthesis and its storage were not as markedly affected by protein deficiency as were those regulating PROTEIN-CALORIE MALNUTRITION IN MALE RATS gonadotrophin release. The concentration of pituitary LH was, in general, similar in both animal groups. Pituitary PRL, on the other hand, tended to be elevated in the controls while FSH was, with one exception, significantly lower in the experimental rats. Other forms of malnutrition have no effect upon pituitary LH, FSH, and PRL (Meites and Reed, '49; Howland, '79, or have been reported to reduce (Meites and Reed, '49; Srebnik and Nelson, '62; Nakanishi et al., '76) or even elevate (Root and Russ, '72; Howland, '75; Nakanishi, '76) pituitary LH, FSH and PRL. Likewise, a variable pattern of LH and FSH secretion by the anterior pituitary gland of malnourished rats was observed in animals challenged by provocative stimuli, such as hemigonadectomy (Stewart et al., '73, '75) and total castration (Srebnik, '70; Root and Russ, '72; Howland and Skinner, '73; Glass et al., '79) or LHRH administration (Root and Duckett, '75; Glass et al., '79). In some instances, the response was greater than that observed in the controltreated animals (Root and Russ, '72; Stewart et al., '75; Glass et al., '791, suggesting that while sufficient levels of gonadotrophins were contained within the pituitary gland which could be released in response to a given challenge, inadequate input from the hypothalamus existed, which over a prolonged period of time, could not maintain normal pituitary function. Our findings of reduced LHRH in the hypothalami of the rats fed the LPD would support this premise. Chronic underfeeding (Piacsek and Meites, '67) and starvation (Negro-Vilar et al., '71) also reduced bioassayable LHRH (Piacsek and Meites, '67) and FSH-RF (Negro-Vilar et al., '71); however, hypothalamic LHRH, a s measured by radioimmunoassay, was unchanged in rats deprived of food for seven days (Root et al., '75). The question then arises as t o why the hypothalamo-hypophyseal-gonadal axis does not give a "castration-response" in the protein-deficient rat. The answer may in part be related to the extrahypothalamic control over reproductive physiology. Pineal gland activity seems to be enhanced by underfeeding (Piacsek and Meites, '67; Sorrentino et al., '71; Walker and Bethea, '77; Walker and Frawley, '77). Exposing malnourished rats to constant light (Piacsek and Meites, '67) or pinealectomizing blinded underfed animals (Sorrentino et al., '71) increased gonadal and sex accessory organ weight. Moreover, a greater compensatory ovarian hypertrophy response (Walker and Bethea. '77) and higher serum LH values (Walker and Frawley, '75) were recorded in rats 353 that were chronically underfed and exposed to constant light or pinealectomized than were found in malnourished, untreated animals. 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