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Ultrastructure of the sertoli and leydig cells in the testes of normal versus protein-calorie malnourished rats
NUTRITION RESEARCH, Vol. 3, pp. 61-68, 1983 0271-5317/83/010061-08503.00/0 Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All r i g h t s reserved.
ULTRASTRUCTURE OF THE SERTOLI AND LEYDIG CELLS IN THE TESTES OF NORMAL VERSUS PROTEIN-CALORIE MALNOURISHED RATS Damon C. Herbert, Ph.D. and Frank J. Weaker, Ph.D. Department of Anatomy The University of Texas Health Science Center at San Antonio San Antonio, Texas 78284
ABSTRACT
The morphology of the gonads was examined at the ultrastructural level in 20 to 55 day-old male rats that were protein-calorie malnourished and the results compared to animals fed a standard laboratory diet. The conditions of undernutrition led to a reduction in the growth of the seminiferous tubules. By day 55, the tubules had only developed to a size corresponding to that found in a normal animal legs than 30 days of age. A similar retardation was noted in the maturation of the Sertoli and Leydig cells, although the effect of undernutrition on their morphology was evident at an earlier age in the latter cell type. These findings support our previous studies in which it was reported that dietary deficiencies in proteins and calories lead to a prolongation in the time necessary for sexual maturation to occur in male rats.
Key Words:
protein-calorie malnutrition, cells, electron microscopy
testes, Sertoli cells, Leydig
INTRODUCTION
Protein-calorie (-energy) malnutrition (PCM) " .... continues to be a major public health problem in many developing countries" (i). It has been estimated that up to 90% of preschool children residing in the poorer nations of the wo~id suffer from some form of malnutrition (I). In spite of these data, little information is available which describes the hormonal status of the reproductive system of either preschoolers or adolescents whose diets are deficient in proteins, calories or both (2-4). Recently, our laboratory has examined the changes induced by PCM on the maturation of the testes and of the hypothalamic-hypophyseal-gonadal axis in rats between 20 and 50 days of age in an effort to understand the deleterious effects on the reproductive system brought about by undernutrition (5). In these studies, serum levels of the gonadotrophins, prolactin and testosterone in the malnourished rats were well below those observed in the well-fed con61
62
D.C. Herbert and F.J. Weaker
trois. Epididymal concentrations of androgen binding protein were also reduced (6). Evidence of sexual maturation and puberty onset was noted in only 12.5% of the experimental animals (5). Because of this, it was of interest to examine the ultrastructure of the gonads of rats with PCM to determine the degree to which the morphology of the seminiferous tubules and the interstitium was affected. The detailed results of these studies are contained within the following report.
MATERIALS AND METHODS
A group of thirty-three, 20 day-old Sprague-Dawley male rats were purchased from Simonsen Laboratories, Gilroy, California. They were housed in air conditioned quarters maintained at 22-24~ with the humidity regulated at 50% and the light:dark cycle controlled for 14:10. Eighteen rats were fed a standard laboratory diet (SLD) composed of 27% protein, 59% starch, 10% vegetable oil and 4% salt mixture (USP-XIV), while fifteen animals were given a low protein diet (LPD) containing 8% protein, 78% starch, 10% vegetable oil and 4% salt mixture (USP-XIV). The caloric values of the two diets were 4.3 Kcal per gram and 4.5 Kcal per gram, respectively. Both the SLD and the LPD contained a special vitamin diet fortification mixture prepared by the manufacturer, ICN Nutritional Biochemicals, Cleveland, Ohio. Food and water were provided ad libitum. The changes in the body weight of each animal along with its food consumption were monitored on a daily basis. The latter was computed as previously described (5). One group of animals was sacrificed immediately upon arrival and served as time zero controls. The remaining animals were sacrificed at seven-day intervals from day 27 through day 55. At each interval, three controls and three malnourished animals were anesthetized with Nembutal. The testes of each rat were initially fixed by whole body vascular perfusion, using a ventricular catheter, with a mixture of 4% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer, pH 7.2. The gonads were then removed, diced into I mm cubes and placed into fresh perfusate for 2 hours. Following this, the tissues were post-fixed for 2 hours in 1% osmium tetroxide in 0.1M cacodylate buffer, pH 7.4, rinsed with distilled water and en bloc stained overnight in a 0.5% aqueous solution of uranyl acetate. They were then processed using routine ultrastructural procedures and embedded in Spurr's low viscosity embedding medium. Two ~m thick sections along with the ultrathin sections were obtained using a Reichert OM-U3 ultramicrotome. The thick sections were stained with I% toluidine blue in i% borax and examined using a Zeiss light microscope equipped with an ocular micrometer. The diameters of the seminiferous tubules from an average of 35 tubules per animal were obtained, along with the nuclear diameters from a minimum of 35 Leydig cells per rat. It was technically not possible for us to compute the nuclear size of the Sertoli cells due to their well-known pleomorphism. The data (mean • standard error of the mean) were arranged according to experimental grouping and statistically analyzed by analysis of variance followed by Duncan's multiple range test. The ultrathin sections were mounted on copper grids, stained with lead citrate and uranyl acetate, and examined using a Siemens I 0 1 E l m i s k o p transmission electron microscope.
RESULTS
The body weights of the two animal groups were similar at the beginning of the study. At each of the seven day intervals, between 27 and 55 days of age, the controls grew at a more rapid rate than the malnourished rats, so
Testis U1t r a s t r u c t u r e i n Mal n u t r i t i o n
63
that by day 55, there was a 423% difference in the body weights of the two groups of rats (288.4• gms - controls vs. 68.1• gms - malnourished). The daily intake of food, proteins and Kcals was also greater in the well-fed rats which is in agreement with our earlier findings (5). The diameters of the seminiferous tubules in the gonads of the rats fed the SLD increased throughout the entire experimental period (Fig. i). The rate of growth of the tubules was linear until the animals had reached sexual maturation (based on the presence of step 19 spermatids in the germinal epithelium) at which point the growth rate lessened. In contrast, the development of the seminiferous tubules in the malnourished animals was more erratic. The greatest increase in tubule size in these animals was noted between days 34 and 41 which corresponds to the period when serum follicle stimulating hormone (FSH) reaches its peak in the normal, well-fed rat (5). There were no apparent changes in the morphology of the Sertoli cells between days 20 and 41 as a result of the conditions of PCM (Fig. 2). The cytoplasm of these cells contained a modest number of mitochondria along with lipid droplets, profiles of smooth surfaced endoplasmic reticulum and Golgi associated saccules, vesicles and vacuoles. The nuclei were slightly indented and displayed clumps of heterochromatin subjacent to the surrounding envelope. On days 41, 48 and 55, however, the ultrastrncture of the Sertoli cells was visibly different between the two animal groups (Figs. 3 and 4). The cells increased in size at a slower rate in the malnourished rats which caused them to appear more "crowded" when viewed microscopically. In addition, the nuclei retained the clumps of heterochromatin associated with the envelope. These aggregates were less frequently visible in the nuclei of cells from the control rats. In contrast to the Sertoli cells, the effects of PCM were observed at an earlier age in the interstitium. The rate of growth of the Leydig cells, as reflected by changes in the size of the cell nuclei, was less in the rats fed the LPD (Fig. 5). These differences were accompanied by a reduction in the amount of cytoplasm, smooth surfaced endoplasmic reticulum and elements of the Golgi complex, all of which were readily apparent ultrastructurally from days 41 through 55 (Figs. 6 and 7).
DISCUSSION
It is well known that normal development of the gonads in a sexually immature rat is hormone dependent (7,8). In the testes, the Sertoli cell has been reported to be the prime target cell for the pituitary gonadotrophin, FSH (9), while luteinizing hormone (LH) and prolactin (PRL) are known to act upon the Leydig cells to affect testosterone production (I0). The initiation of the first spermatogenic cycle will not proceed normally if the action of LH or FSH is blocked by passive immunization of prepubertal rats with antisera to ovine LH or rat FSH (11). A concomitant decrease in seminiferous tubule diameters also occurs indicating that normal maintenance of the structural integrity of the tubules is dependent upon both hormones. Thus, in rats with PCM where pituitary and serum concentrations of LH and FSH are markedly reduced (5), it is not surprising that the tubule diameters were also well below those present in control rats. Lack of a sufficient trophic effect on the testes by LH and FSH during conditions of PCM contributed to t h e failure of the malnourished rats to undergo puberty at the same time as their age-matched counterparts fed the SLD ( 5 ) , Peripheral levels of the two hormones in the rats with PCM were significantly lower than those found in control animals which were of comparable size and weight ( 5 ) . Normally, as rats mature sexually and approach puberty, there is an increase in the germinal elements of the seminiferous epithelium (12). A simultaneous expansion of the Sertoli cell
64
D.C. Herbert and F.J. Weaker
DIAMETER OF SEMINIFEROUSTUBULES :550]
300FIG. 1 Diameters of the seminiferous tubules of the control animals (closed circles) were significantly greater than those of the malnourished rats (open circles) from days 27 through 55.
F 250s
150- ~
O2o
[p<~20012
14 4, g8
55
Age (days)
FIG. 2 An electron micrograph of a Sertoli cell from a 27 day-old PCM rat. Note the aggregates of nuclear heterochromatin which were characteristic of control and experimental animals between days 27 and 41. 10,000X.
Testis U1trastructure in Mal n u t r i t i o n
65
FIGS. 3 and 4 The Sertoli cells in 48 day-old rats fed the standard laboratory diet (fig. 3) displayed greater cytoplasmic and nuclear growth than what was observed in the gonads of the age-matched malnourished rats (fig. 4). The cells in the latter group of animals resembled those found in prepubertal rats (compare with fig. 2). 7,400X.
66
D.C. Herbert and F.J. Weaker
NUCLEAR DIAMETER OF LEYDIG CELLS
FIG. 5 A more rapid nuclear growth rate was observed in the Leydig cells of the controls (closed circles) as compared to the experimental animals (open circles) throughout the entire course of the study.
E6-
on
OJf
I 27
r 34
I 41
I 48
55
Age (days)
cytoplasm occurs in order to support and nourish the developing germ cells. Since the growth of the germinal epithelium was not as extensive in the malnourished rats, there was less of a physiological need for the Sertoli cells to develop. This led to their more "crowded" ultrastructural appearance. Below normal levels of pituitary LH and FSH are believed to be responsible for this occurrence and for the reduction in the rate at which the cytoplasmic organelles developed along with the presence of nuclear aggregations of heterochromatin which were particularly prominent at an age (i.e., days 48 and 55) when puberty onset should have occurred. Similar ultrastructural phenomena have been shown to be associated with Sertoli cells in rats passively immunized with antisera to the gonadotrophins (13). Even though this cell type was and continued to be morphologically immature throughout the entire course of our study, we failed to detect any effect of PCM on the integrity of the blood-testis barrier. Chemes et al. (13) made the same observation in their animals. The Sertoli cells in protein and calorically deficient animals were not only affected structurally but functionally as well. Their production of androgen binding protein in 50 day-old rats fed a LPD for 30 days was less than 30% of what was secreted by well-fed animals (6). Moreover, the concentrations measured on day 50 were below those observed in control animals that were 25 days of age. The activity of another Sertoli cell protein, inhibin, has also been shown to be subnormal under conditions of PCM (14). Light microscopic examination of the testicular interstitum of the malnourished rats revealed a decrease in both the size and number of Leydig cells (5). The present study provides more quantitative support to this observation. The nuclear diameters along with the development of the smooth surfaced endoplasmic reticulum in the 55 day-old experimental animals resembled that found in prepubertal rats that were fed the SLD. The failure of these organelles to develop at a normal rate is again largely due to the absence of adequate stimulation by the pituitary gonadotrophins, particularly
Testis Ultrastructure in Malnutrition
67
FIGS. 6 and 7 By day 55, the Leydig cells in the well-fed rats (fig. abundance of steroid-synthesizing organelles. In contrast, rather poorly developed in the age-matched experimental
II,IOOX.
6) contained an these cells were rats (fig. 7).
68
D.C. Herbert and F.J. Weaker
LH and PRL (5). As a result, the cells lack the volume of organelles necessary to produce a sufficient quantity of testosterone (5) to participate fully in the processes associated with spermatogenesis or in the normal maturation of steroid target organs such as the prostate gland (5,15). Because of the complexities of the physiologic actions of the gonadotrophins and of the sex steroids (16), the conditions of hypopituiturism and hypogonadism precipitated by PCM result in rather extensive effects on the reproductive axis (5,6,14,). Taken together, these actions contribute to the delay in puberty onset and the continued immature state which is displayed by the hypothalamo-hypophyseal-gonadal axis in rats that are protein and calorically deficient.
ACKNOWLEDGMENTS
This work was supported by grants from the NIH (HD 10914 and P30 HDI0202) and NSF (PCM 8118487). The authors wish to thank Ms. Donna Kenneally for her expert technical assistance.
REFERENCES
i.
REDDY, V. Protein energy malnutrition; an overview. In: Nutrition in Health and Disease and International Development. A.E. Harper and G. K. Davis (eds). A.R. Liss, Inc., New York, 1981, pp. 227-235.
2.
PIMSTONE, B. Endocrine function in protein-calorie malnutrition. Clin. Endocrinol. 5: 79-95, 1976.
3.
PIMSTONE, B., BECKER, D.J. and KRONHEIM, S. FSH and LH response to LH releasing hormone in normal and malnourished infants. Horm. Metab. Res., Suppl. ~: 179-184, 1974.
4.
BECKER, D.J., VINIK, A.I., PIMSTONE, B. and PAUL, M. Prolactin responses to thyrotropin-releasing hormone in protein-calorie malnutrition. J. Clin. Endocrinol. Metab. 41: 782-783, 1975.
5.
HERBERT, D.C. Growth patterns and hormonal profile of male rats with protein-calorie malnutrition. Anat. Res. 197: 339-354, 1980.
6.
HERBERT, D.C. and TINDALL, D.J. Epididymal androgen binding protein in protein-calorie malnourished rats. Endocr. Res. Com~un. 7: 61-70, 1980.
7.
DEJONG, F.H. and SHARPE, R.M. The onset and establishment of spermatogenesis in rats in relation to gonadotrophin and testosterone levels. J. Endocrinol. 75: 197-207, 1977.
8.
NAZIAN, S.J. and MAHESH, V.B. Hypothalamic, pituitary, testicular, and secondary organ functions and interactions during the sexual maturation of the male rat. Arch. Androl. 4: 283-303, 1980.
9.
RITZEN, E.M., HANSSON, V. and FRENCH, F.S. The Sertoli cell. In: The Testis. H. Burger and D. de Kretser (eds). Raven Press, New York, 1981, pp. 171-194.
ULTRASTRUCTURE OF THE SERTOLI AND LEYDIG CELLS IN THE TESTES OF NORMAL VERSUS PROTEIN-CALORIE MALNOURISHED RATS Damon C. Herbert, Ph.D. and Frank J. Weaker, Ph.D. Department of Anatomy The University of Texas Health Science Center at San Antonio San Antonio, Texas 78284
ABSTRACT
The morphology of the gonads was examined at the ultrastructural level in 20 to 55 day-old male rats that were protein-calorie malnourished and the results compared to animals fed a standard laboratory diet. The conditions of undernutrition led to a reduction in the growth of the seminiferous tubules. By day 55, the tubules had only developed to a size corresponding to that found in a normal animal legs than 30 days of age. A similar retardation was noted in the maturation of the Sertoli and Leydig cells, although the effect of undernutrition on their morphology was evident at an earlier age in the latter cell type. These findings support our previous studies in which it was reported that dietary deficiencies in proteins and calories lead to a prolongation in the time necessary for sexual maturation to occur in male rats.
Key Words:
protein-calorie malnutrition, cells, electron microscopy
testes, Sertoli cells, Leydig
INTRODUCTION
Protein-calorie (-energy) malnutrition (PCM) " .... continues to be a major public health problem in many developing countries" (i). It has been estimated that up to 90% of preschool children residing in the poorer nations of the wo~id suffer from some form of malnutrition (I). In spite of these data, little information is available which describes the hormonal status of the reproductive system of either preschoolers or adolescents whose diets are deficient in proteins, calories or both (2-4). Recently, our laboratory has examined the changes induced by PCM on the maturation of the testes and of the hypothalamic-hypophyseal-gonadal axis in rats between 20 and 50 days of age in an effort to understand the deleterious effects on the reproductive system brought about by undernutrition (5). In these studies, serum levels of the gonadotrophins, prolactin and testosterone in the malnourished rats were well below those observed in the well-fed con61
62
D.C. Herbert and F.J. Weaker
trois. Epididymal concentrations of androgen binding protein were also reduced (6). Evidence of sexual maturation and puberty onset was noted in only 12.5% of the experimental animals (5). Because of this, it was of interest to examine the ultrastructure of the gonads of rats with PCM to determine the degree to which the morphology of the seminiferous tubules and the interstitium was affected. The detailed results of these studies are contained within the following report.
MATERIALS AND METHODS
A group of thirty-three, 20 day-old Sprague-Dawley male rats were purchased from Simonsen Laboratories, Gilroy, California. They were housed in air conditioned quarters maintained at 22-24~ with the humidity regulated at 50% and the light:dark cycle controlled for 14:10. Eighteen rats were fed a standard laboratory diet (SLD) composed of 27% protein, 59% starch, 10% vegetable oil and 4% salt mixture (USP-XIV), while fifteen animals were given a low protein diet (LPD) containing 8% protein, 78% starch, 10% vegetable oil and 4% salt mixture (USP-XIV). The caloric values of the two diets were 4.3 Kcal per gram and 4.5 Kcal per gram, respectively. Both the SLD and the LPD contained a special vitamin diet fortification mixture prepared by the manufacturer, ICN Nutritional Biochemicals, Cleveland, Ohio. Food and water were provided ad libitum. The changes in the body weight of each animal along with its food consumption were monitored on a daily basis. The latter was computed as previously described (5). One group of animals was sacrificed immediately upon arrival and served as time zero controls. The remaining animals were sacrificed at seven-day intervals from day 27 through day 55. At each interval, three controls and three malnourished animals were anesthetized with Nembutal. The testes of each rat were initially fixed by whole body vascular perfusion, using a ventricular catheter, with a mixture of 4% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer, pH 7.2. The gonads were then removed, diced into I mm cubes and placed into fresh perfusate for 2 hours. Following this, the tissues were post-fixed for 2 hours in 1% osmium tetroxide in 0.1M cacodylate buffer, pH 7.4, rinsed with distilled water and en bloc stained overnight in a 0.5% aqueous solution of uranyl acetate. They were then processed using routine ultrastructural procedures and embedded in Spurr's low viscosity embedding medium. Two ~m thick sections along with the ultrathin sections were obtained using a Reichert OM-U3 ultramicrotome. The thick sections were stained with I% toluidine blue in i% borax and examined using a Zeiss light microscope equipped with an ocular micrometer. The diameters of the seminiferous tubules from an average of 35 tubules per animal were obtained, along with the nuclear diameters from a minimum of 35 Leydig cells per rat. It was technically not possible for us to compute the nuclear size of the Sertoli cells due to their well-known pleomorphism. The data (mean • standard error of the mean) were arranged according to experimental grouping and statistically analyzed by analysis of variance followed by Duncan's multiple range test. The ultrathin sections were mounted on copper grids, stained with lead citrate and uranyl acetate, and examined using a Siemens I 0 1 E l m i s k o p transmission electron microscope.
RESULTS
The body weights of the two animal groups were similar at the beginning of the study. At each of the seven day intervals, between 27 and 55 days of age, the controls grew at a more rapid rate than the malnourished rats, so
Testis U1t r a s t r u c t u r e i n Mal n u t r i t i o n
63
that by day 55, there was a 423% difference in the body weights of the two groups of rats (288.4• gms - controls vs. 68.1• gms - malnourished). The daily intake of food, proteins and Kcals was also greater in the well-fed rats which is in agreement with our earlier findings (5). The diameters of the seminiferous tubules in the gonads of the rats fed the SLD increased throughout the entire experimental period (Fig. i). The rate of growth of the tubules was linear until the animals had reached sexual maturation (based on the presence of step 19 spermatids in the germinal epithelium) at which point the growth rate lessened. In contrast, the development of the seminiferous tubules in the malnourished animals was more erratic. The greatest increase in tubule size in these animals was noted between days 34 and 41 which corresponds to the period when serum follicle stimulating hormone (FSH) reaches its peak in the normal, well-fed rat (5). There were no apparent changes in the morphology of the Sertoli cells between days 20 and 41 as a result of the conditions of PCM (Fig. 2). The cytoplasm of these cells contained a modest number of mitochondria along with lipid droplets, profiles of smooth surfaced endoplasmic reticulum and Golgi associated saccules, vesicles and vacuoles. The nuclei were slightly indented and displayed clumps of heterochromatin subjacent to the surrounding envelope. On days 41, 48 and 55, however, the ultrastrncture of the Sertoli cells was visibly different between the two animal groups (Figs. 3 and 4). The cells increased in size at a slower rate in the malnourished rats which caused them to appear more "crowded" when viewed microscopically. In addition, the nuclei retained the clumps of heterochromatin associated with the envelope. These aggregates were less frequently visible in the nuclei of cells from the control rats. In contrast to the Sertoli cells, the effects of PCM were observed at an earlier age in the interstitium. The rate of growth of the Leydig cells, as reflected by changes in the size of the cell nuclei, was less in the rats fed the LPD (Fig. 5). These differences were accompanied by a reduction in the amount of cytoplasm, smooth surfaced endoplasmic reticulum and elements of the Golgi complex, all of which were readily apparent ultrastructurally from days 41 through 55 (Figs. 6 and 7).
DISCUSSION
It is well known that normal development of the gonads in a sexually immature rat is hormone dependent (7,8). In the testes, the Sertoli cell has been reported to be the prime target cell for the pituitary gonadotrophin, FSH (9), while luteinizing hormone (LH) and prolactin (PRL) are known to act upon the Leydig cells to affect testosterone production (I0). The initiation of the first spermatogenic cycle will not proceed normally if the action of LH or FSH is blocked by passive immunization of prepubertal rats with antisera to ovine LH or rat FSH (11). A concomitant decrease in seminiferous tubule diameters also occurs indicating that normal maintenance of the structural integrity of the tubules is dependent upon both hormones. Thus, in rats with PCM where pituitary and serum concentrations of LH and FSH are markedly reduced (5), it is not surprising that the tubule diameters were also well below those present in control rats. Lack of a sufficient trophic effect on the testes by LH and FSH during conditions of PCM contributed to t h e failure of the malnourished rats to undergo puberty at the same time as their age-matched counterparts fed the SLD ( 5 ) , Peripheral levels of the two hormones in the rats with PCM were significantly lower than those found in control animals which were of comparable size and weight ( 5 ) . Normally, as rats mature sexually and approach puberty, there is an increase in the germinal elements of the seminiferous epithelium (12). A simultaneous expansion of the Sertoli cell
64
D.C. Herbert and F.J. Weaker
DIAMETER OF SEMINIFEROUSTUBULES :550]
300FIG. 1 Diameters of the seminiferous tubules of the control animals (closed circles) were significantly greater than those of the malnourished rats (open circles) from days 27 through 55.
F 250s
150- ~
O2o
[p<~20012
14 4, g8
55
Age (days)
FIG. 2 An electron micrograph of a Sertoli cell from a 27 day-old PCM rat. Note the aggregates of nuclear heterochromatin which were characteristic of control and experimental animals between days 27 and 41. 10,000X.
Testis U1trastructure in Mal n u t r i t i o n
65
FIGS. 3 and 4 The Sertoli cells in 48 day-old rats fed the standard laboratory diet (fig. 3) displayed greater cytoplasmic and nuclear growth than what was observed in the gonads of the age-matched malnourished rats (fig. 4). The cells in the latter group of animals resembled those found in prepubertal rats (compare with fig. 2). 7,400X.
66
D.C. Herbert and F.J. Weaker
NUCLEAR DIAMETER OF LEYDIG CELLS
FIG. 5 A more rapid nuclear growth rate was observed in the Leydig cells of the controls (closed circles) as compared to the experimental animals (open circles) throughout the entire course of the study.
E6-
on
OJf
I 27
r 34
I 41
I 48
55
Age (days)
cytoplasm occurs in order to support and nourish the developing germ cells. Since the growth of the germinal epithelium was not as extensive in the malnourished rats, there was less of a physiological need for the Sertoli cells to develop. This led to their more "crowded" ultrastructural appearance. Below normal levels of pituitary LH and FSH are believed to be responsible for this occurrence and for the reduction in the rate at which the cytoplasmic organelles developed along with the presence of nuclear aggregations of heterochromatin which were particularly prominent at an age (i.e., days 48 and 55) when puberty onset should have occurred. Similar ultrastructural phenomena have been shown to be associated with Sertoli cells in rats passively immunized with antisera to the gonadotrophins (13). Even though this cell type was and continued to be morphologically immature throughout the entire course of our study, we failed to detect any effect of PCM on the integrity of the blood-testis barrier. Chemes et al. (13) made the same observation in their animals. The Sertoli cells in protein and calorically deficient animals were not only affected structurally but functionally as well. Their production of androgen binding protein in 50 day-old rats fed a LPD for 30 days was less than 30% of what was secreted by well-fed animals (6). Moreover, the concentrations measured on day 50 were below those observed in control animals that were 25 days of age. The activity of another Sertoli cell protein, inhibin, has also been shown to be subnormal under conditions of PCM (14). Light microscopic examination of the testicular interstitum of the malnourished rats revealed a decrease in both the size and number of Leydig cells (5). The present study provides more quantitative support to this observation. The nuclear diameters along with the development of the smooth surfaced endoplasmic reticulum in the 55 day-old experimental animals resembled that found in prepubertal rats that were fed the SLD. The failure of these organelles to develop at a normal rate is again largely due to the absence of adequate stimulation by the pituitary gonadotrophins, particularly
Testis Ultrastructure in Malnutrition
67
FIGS. 6 and 7 By day 55, the Leydig cells in the well-fed rats (fig. abundance of steroid-synthesizing organelles. In contrast, rather poorly developed in the age-matched experimental
II,IOOX.
6) contained an these cells were rats (fig. 7).
68
D.C. Herbert and F.J. Weaker
LH and PRL (5). As a result, the cells lack the volume of organelles necessary to produce a sufficient quantity of testosterone (5) to participate fully in the processes associated with spermatogenesis or in the normal maturation of steroid target organs such as the prostate gland (5,15). Because of the complexities of the physiologic actions of the gonadotrophins and of the sex steroids (16), the conditions of hypopituiturism and hypogonadism precipitated by PCM result in rather extensive effects on the reproductive axis (5,6,14,). Taken together, these actions contribute to the delay in puberty onset and the continued immature state which is displayed by the hypothalamo-hypophyseal-gonadal axis in rats that are protein and calorically deficient.
ACKNOWLEDGMENTS
This work was supported by grants from the NIH (HD 10914 and P30 HDI0202) and NSF (PCM 8118487). The authors wish to thank Ms. Donna Kenneally for her expert technical assistance.
REFERENCES
i.
REDDY, V. Protein energy malnutrition; an overview. In: Nutrition in Health and Disease and International Development. A.E. Harper and G. K. Davis (eds). A.R. Liss, Inc., New York, 1981, pp. 227-235.
2.
PIMSTONE, B. Endocrine function in protein-calorie malnutrition. Clin. Endocrinol. 5: 79-95, 1976.
3.
PIMSTONE, B., BECKER, D.J. and KRONHEIM, S. FSH and LH response to LH releasing hormone in normal and malnourished infants. Horm. Metab. Res., Suppl. ~: 179-184, 1974.
4.
BECKER, D.J., VINIK, A.I., PIMSTONE, B. and PAUL, M. Prolactin responses to thyrotropin-releasing hormone in protein-calorie malnutrition. J. Clin. Endocrinol. Metab. 41: 782-783, 1975.
5.
HERBERT, D.C. Growth patterns and hormonal profile of male rats with protein-calorie malnutrition. Anat. Res. 197: 339-354, 1980.
6.
HERBERT, D.C. and TINDALL, D.J. Epididymal androgen binding protein in protein-calorie malnourished rats. Endocr. Res. Com~un. 7: 61-70, 1980.
7.
DEJONG, F.H. and SHARPE, R.M. The onset and establishment of spermatogenesis in rats in relation to gonadotrophin and testosterone levels. J. Endocrinol. 75: 197-207, 1977.
8.
NAZIAN, S.J. and MAHESH, V.B. Hypothalamic, pituitary, testicular, and secondary organ functions and interactions during the sexual maturation of the male rat. Arch. Androl. 4: 283-303, 1980.
9.
RITZEN, E.M., HANSSON, V. and FRENCH, F.S. The Sertoli cell. In: The Testis. H. Burger and D. de Kretser (eds). Raven Press, New York, 1981, pp. 171-194.