9 1990 by the Humana Press, Inc. All rights of any nature, whatsoever, reserved. 0163-4984/90/2401-0001 $02.20
Zinc Deficiency and Dipeptidyl Carboxypeptidase ActMty Comparative Effects on Epididymis and Testis of Rats PHILIP G. REEVES Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center, PO Box 7166, University Station, Grand Forks, ND 58202-7166 Received November 15, 1988; Accepted February 23, 1989
ABSTRACT Dipeptidyl carboxypeptidase (DC) is highly active in the testis and epididymis of rats and increases during pubertal development. Zinc deficiency during this period depresses the activity of DC in the testis. Experiments were conducted to determine the effects of zinc deficiency on epididymal DC activity. Comparisons were made between changes seen in this organ and those observed in testis. Three dietary treatments were used; zinc-deficient, fed ad libitum; zincadequate, pair-fed to the deficient group; and zinc-adequate, fed ad libitum. Results confirmed that testicular DC is affected negatively by zinc deficiency. DC activity was also lower in the epididymis of zincdeficient rats than in control rats. These effects apparently were specific relative to changes in activity of other enzymes. Alkaline phosphatase activity in the epididymis was not affected by zinc deficiency and it was depressed in the testis. Gamma-glytamyl transPresented in part at the 1988 Joint Meeting of the North Dakota and South Dakota Academies of Science, Bismarck, ND, April 30, 1988. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.
Biological Trace Element Research
]
Vol. 24, 1990
Reeves
2
ferase activity in the epididymis was not affected by zinc deficiency but it was elevated in the testis. The results of this study suggest that part of the effect of zinc deficiency on sexual maturity in the male rat may be caused by reduced activity of DC. This enzyme is thought to be required for maturation and development of sperm cells. Index Entries: Angiotensin converting enzyme; ACE; alkaline phosphatase; gamma-glutamyl transferase; rats; reproduction.
INTRODUCTION Zinc deficiency in animals causes a variety of physiological abnormalities. One of major importance is an inhibition of sexual maturation in the male. The deficiency leads to damage of the seminiferous tubules and a reduction in the number of spermatozoa. In addition, underdevelopment of both primary and secondary sex characteristics occurs (1,2). Dipeptidyl carboxypeptidase (angiotensin converting enzyme, peptidyldipeptide hydrolase, EC 3.4.15.1, DC) was first shown by Cushman and Cheung (3) to be highly active in the testis and epididymis of rats. There is a marked increase in DC activity during the differentiation of these tissues at puberty (4). Lung DC is known to be a zinc metalloenzyme (5,6) and the testicular enzyme is thought to be also. Recent studies have shown that the activity of this enzyme is severely depressed in zinc-deficient rats (7). The epididymis of the rat contains two isoenzymes of DC with relatively high activity (8). Their catalytic activities are similar but the immunochemical characteristics of one corresponds to lung DC, whereas the other is more like that of testes DC (9,10). Because no one had yet shown that epididymal DC activity was affected by zinc deficiency, it became the main objective of this study to determine if there were an effect. The second objective was to compare changes observed in the epididymis with those in the testis. In this study, no distinction was made between the epididymal isoenzymes. Hence, I shall refer to them collectively as epididymal dipeptidyl carboxypeptidase, or EDC, throughout the text. Bettger and O'Dell (11) discussed the importance of zinc in the structure and function of cellular membranes. The particulate portion of the testes contains more than 95% of total TDC activity, whereas the particulate portion of the epididymis contains only about 70% of total EDC activity (9). To determine the specificity of the effect of zinc status on the activity of DC in these tissues, the activities of other membranebound enzymes were determined. I chose alkaline phosphatase, which is a zinc dependent enzyme and gamma-glutamyl transferase, not known to be zinc dependent.
Biological Trace Element Research
Vol. 24, 1990
Zinc and Dipeptidyl Activity
3 Table 1
Composition of the Basal Diet
Ingredients
g/kg
Corn starch Glucose h y d r a t e Dried egg white ~ Corn oiP Soybean oil '~ Modified AIN-76 mineral premix d Cellulose e AIN-76 vitamin premix r Choline premixg Biotin premix ~
330 300 175 50 50 35 30 10 10 10
aTeklad, Madison, WI, Cat. #160230. ~Mazola Oil, Best Foods CPC International, Inc., Englewood Cliffs, NJ. cCrisco Oil, Procter & Gamble, Cincinnati, OH. aMineral Premix (Teklad, Madison, WI, Cat. #TD87282) provided the following ingredients in g per kg of diet: CaHPO4, 17.5; K3C6HsO7 9 H20 (potassium citrate), 7.7; K2SO4, 1.82; NaC1, 1.092; MgO, 0.84; ferric citrate (17% Fe), 0.21; MnCO3, 0.1225; CrK(SO4)2 9 12H20, 0.01925; CuCO3, 0.0105; KI, 0.00035; Na2SeO 3 9 5H20, 0.00035. The mineral mix provided 0.43 g of Na per kg of diet and dried egg white provided approximately 1.7 g per kg of diet, for a total Na content of 2.13 g per kg diet. ZnCO 3 was used as the source of Zn in the supplemented diet. q~eklad, Madison, WI, Cat. #160390. rFeklad, Madison, WI, Cat. #40077. ,250 g of choline bitartrate per kg in finely powdered dextrose. ~80 mg of d-biotin per kg in finely powdered dextrose.
MATERIALS AND METHODS
Expedmental Protocol Twenty male Long-Evans rats (Harland, Madison, WI), approximately 4 wk old, were divided into 4 groups of 5 rats each. One group was fed the basal diet (Table 1) that contained
Zinc Deficiency and Dipeptidyl Carboxypeptidase ActMty Comparative Effects on Epididymis and Testis of Rats PHILIP G. REEVES Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center, PO Box 7166, University Station, Grand Forks, ND 58202-7166 Received November 15, 1988; Accepted February 23, 1989
ABSTRACT Dipeptidyl carboxypeptidase (DC) is highly active in the testis and epididymis of rats and increases during pubertal development. Zinc deficiency during this period depresses the activity of DC in the testis. Experiments were conducted to determine the effects of zinc deficiency on epididymal DC activity. Comparisons were made between changes seen in this organ and those observed in testis. Three dietary treatments were used; zinc-deficient, fed ad libitum; zincadequate, pair-fed to the deficient group; and zinc-adequate, fed ad libitum. Results confirmed that testicular DC is affected negatively by zinc deficiency. DC activity was also lower in the epididymis of zincdeficient rats than in control rats. These effects apparently were specific relative to changes in activity of other enzymes. Alkaline phosphatase activity in the epididymis was not affected by zinc deficiency and it was depressed in the testis. Gamma-glytamyl transPresented in part at the 1988 Joint Meeting of the North Dakota and South Dakota Academies of Science, Bismarck, ND, April 30, 1988. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.
Biological Trace Element Research
]
Vol. 24, 1990
Reeves
2
ferase activity in the epididymis was not affected by zinc deficiency but it was elevated in the testis. The results of this study suggest that part of the effect of zinc deficiency on sexual maturity in the male rat may be caused by reduced activity of DC. This enzyme is thought to be required for maturation and development of sperm cells. Index Entries: Angiotensin converting enzyme; ACE; alkaline phosphatase; gamma-glutamyl transferase; rats; reproduction.
INTRODUCTION Zinc deficiency in animals causes a variety of physiological abnormalities. One of major importance is an inhibition of sexual maturation in the male. The deficiency leads to damage of the seminiferous tubules and a reduction in the number of spermatozoa. In addition, underdevelopment of both primary and secondary sex characteristics occurs (1,2). Dipeptidyl carboxypeptidase (angiotensin converting enzyme, peptidyldipeptide hydrolase, EC 3.4.15.1, DC) was first shown by Cushman and Cheung (3) to be highly active in the testis and epididymis of rats. There is a marked increase in DC activity during the differentiation of these tissues at puberty (4). Lung DC is known to be a zinc metalloenzyme (5,6) and the testicular enzyme is thought to be also. Recent studies have shown that the activity of this enzyme is severely depressed in zinc-deficient rats (7). The epididymis of the rat contains two isoenzymes of DC with relatively high activity (8). Their catalytic activities are similar but the immunochemical characteristics of one corresponds to lung DC, whereas the other is more like that of testes DC (9,10). Because no one had yet shown that epididymal DC activity was affected by zinc deficiency, it became the main objective of this study to determine if there were an effect. The second objective was to compare changes observed in the epididymis with those in the testis. In this study, no distinction was made between the epididymal isoenzymes. Hence, I shall refer to them collectively as epididymal dipeptidyl carboxypeptidase, or EDC, throughout the text. Bettger and O'Dell (11) discussed the importance of zinc in the structure and function of cellular membranes. The particulate portion of the testes contains more than 95% of total TDC activity, whereas the particulate portion of the epididymis contains only about 70% of total EDC activity (9). To determine the specificity of the effect of zinc status on the activity of DC in these tissues, the activities of other membranebound enzymes were determined. I chose alkaline phosphatase, which is a zinc dependent enzyme and gamma-glutamyl transferase, not known to be zinc dependent.
Biological Trace Element Research
Vol. 24, 1990
Zinc and Dipeptidyl Activity
3 Table 1
Composition of the Basal Diet
Ingredients
g/kg
Corn starch Glucose h y d r a t e Dried egg white ~ Corn oiP Soybean oil '~ Modified AIN-76 mineral premix d Cellulose e AIN-76 vitamin premix r Choline premixg Biotin premix ~
330 300 175 50 50 35 30 10 10 10
aTeklad, Madison, WI, Cat. #160230. ~Mazola Oil, Best Foods CPC International, Inc., Englewood Cliffs, NJ. cCrisco Oil, Procter & Gamble, Cincinnati, OH. aMineral Premix (Teklad, Madison, WI, Cat. #TD87282) provided the following ingredients in g per kg of diet: CaHPO4, 17.5; K3C6HsO7 9 H20 (potassium citrate), 7.7; K2SO4, 1.82; NaC1, 1.092; MgO, 0.84; ferric citrate (17% Fe), 0.21; MnCO3, 0.1225; CrK(SO4)2 9 12H20, 0.01925; CuCO3, 0.0105; KI, 0.00035; Na2SeO 3 9 5H20, 0.00035. The mineral mix provided 0.43 g of Na per kg of diet and dried egg white provided approximately 1.7 g per kg of diet, for a total Na content of 2.13 g per kg diet. ZnCO 3 was used as the source of Zn in the supplemented diet. q~eklad, Madison, WI, Cat. #160390. rFeklad, Madison, WI, Cat. #40077. ,250 g of choline bitartrate per kg in finely powdered dextrose. ~80 mg of d-biotin per kg in finely powdered dextrose.
MATERIALS AND METHODS
Expedmental Protocol Twenty male Long-Evans rats (Harland, Madison, WI), approximately 4 wk old, were divided into 4 groups of 5 rats each. One group was fed the basal diet (Table 1) that contained
