0013-7227/78/1033-0748$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 103, No. 3 Printed in U.S.A.
Effect of Hypophysectomy of Cholesteryl Ester Synthesis and Hydrolysis in Testes and on Serum LecithinCholesterol Acyltransferase Activity in Rats* TAKEHIKO TAKATORIf AND 0. S. PRIVETT The Hormel Institute, University of Minnesota, Austin, Minnesota 55912 ABSTRACT. Studies are reported of the effect of hypophysectomy on cholesterol esterase activity of testicular tissue and serum lecithin-cholesterol acyltransferase (LC AT) activity in rats. The testes of male SpragueDawley rats of 200-255 g were excised from animals sacrificed at 3, 7, and 15 days after hypophysectomy. Assays for cholesterol-esterifying and hydrolytic activities of the testicular tissues of these animals, compared to control animals, showed that hypophysectomy decreased both cholesteryl ester synthesis and hydrolysis.
Hydrolytic activity was affected to a greater extent than esterifying activity. LCAT activity was significantly decreased by hypophysectomy compared to that of control animals. Although serum LCAT and testicular cholesterol esterase activities were decreased, the overall effect of hypophysectomy produced an increase in the level of serum cholesterol and cholesteryl esters. It is suggested that the role of essential fatty acids (EFA) in testicular
function is related to the utilization of cholesteryl esters in androgen synthesis. (Endocrinology 103: 748, 1978)
H
YPOPHYSECTOMY has a pronounced effect on the testes, producing large changes in the level and composition of the lipids (1-7), particularly cholesteryl esters (5-7). Among the changes in cholesteryl esters after hypophysectomy of mature rats is a large elevation of docosapentaenoic acid (7, 8), which is the major EFA in testes. In spite of the importance of EFA in testes development and spermatogenesis (9-15), the physiological role of these fatty acids in testicular function has not been elucidated. It has been shown that the mobilization of cholesteryl esters and the cholesterol fraction of these compounds are directly involved in androgen synthesis, which is under hormonal regulation of the pituitary (16-19). The studies of Behrman and Armstrong (20) and Behrman and Greep (21) showed that cholesterol esterase activity in ovarian tissue is increased upon LH treatReceived June 27, 1977. Address requests for reprints to: Dr. O. S. Privett, The Hormel Institute, University of Minnesota, 801 16th Avenue N.E., Austin, Minnesota 55912. * This work was supported in part by USPHS Grants AM-04942 from the NIH and HL-08214 from the Program Project Branch, Extramural Programs, the National Heart Institute, and the Hormel Foundation. f Present address: Department of Legal Medicine, Hokkaido University School of Medicine, Sapporo 060, Japan.
ment, suggesting that the activity of the enzyme system is a site of the regulation of steroidogenesis. Moreover, cholesterol esterase seemed to exhibit a selectivity for the hydrolysis of unsaturated cholesteryl esters (21). In a previous study (22), we investigated the enzymatic properties of cholesteryl ester synthesis and hydrolysis in rat testes. In the present study, the effect of hypophysectomy on enzyme activity in the synthesis and hydrolysis of cholesteryl esters in testes and on serum lecithin-cholesterol acyltransferase (LCAT) activity in rats is reported. Materials and Methods Animals Hypophysectomized and control male rats of the Sprague-Dawley strain, 200-225 g BW, were obtained from the Hormone Assay Laboratory (Chicago, IL), and fed Purina lab chow ad libitum. Groups of six hypophysectomized and five control animals were sacrificed at 3, 7, and 15 days after hypophysectomy. Enzyme preparation The animals were killed by decapitation and the testes were excised immediately. After removal of the capsule and visible blood vessels, each testis
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The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 19:26 For personal use only. No other uses without permission. . All rights reserved.
CHOLESTEROL ESTERIFICATION IN RAT SERUM AND TESTES was minced and then homogenized in 5 ml 0.25 M sucrose using a Potter-Elvehjem homogenizer, as previously described (22). Cell debris and nuclei were separated from the homogenates by centrifugation at 750 X g for 15 min, which provided a supernatant as the enzyme source. All procedures were carried out at 0 C and protein content of the enzyme preparations was determined by the Lowry method (23) using bovine serum albumin as a standard.
749
nanomoles of cholesterol esterified per min per liter of serum at 37 C by multiplying the percentage of labeled cholesterol esterified by the free cholesterol of the original sample. Significance of the data was determined by Student's t test.
Results
There was a large decrease in the size of testes of the hypophysectomized animals, as shown in Table 1. The effect of hypophysecSubstrates and other chemicals tomy on testicular cholesteryl ester synthesis [4-14C]Cholesterol (SA, 52.0 mCi/mmol) and [4- and hydrolysis are also shown in Table 1. M C]cholesteryl linoleate (SA, 20.9 mCi/mmol) were Seven days after hyophysectomy, cholesterylpurchased from New England Nuclear Corp. (Bos- esterifying activity was significantly decreased ton, MA). Impurities were separated from these compared to that of the control group. The preparations by thin layer chromatography (TLC) cholesteryl ester hydrolyzing activity started using plates coated with silica gel H and a solvent to show the effect of hypophysectomy at this system of petroleum ether-diethyl ether-acetic acid time and was significantly lower than that of (95:15:1, vol/vol). the control group 15 days after hypophysecBovine serum albumin (fraction V) was pur- tomy. In fact, hydrolyzing activity was inchased from Sigma Chemical Co. (St. Louis, MO), hibited to a greater extent than esterifying and cholesterol and cholesteryl linoleate were obactivity in the 15-day posthypophysectomized tained from the Lipids Preparation Laboratory of the Hormel Institute (Austin, MN). Scintillation group, inasmuch as the ratio of the activities chemicals were obtained from Packard Instrument was only approximately 1:1 compared to 2:1 in the controls. Co. (Downers Grove, IL). Hypophysectomy also caused a decrease in Cholesterol-esterifying and cholesteryl ester - LCAT activity within 7 days, as shown in hydrolyzing activities Table 2. Serum cholesterol was elevated 3 The incubation systems and analytical proce- days after hypophysectomy and remained dures were carried out, as previously described (22), high; the level of the cholesteryl esters inin which optimal conditions for esterification and creased more gradually and became signifihydrolysis were defined. Concentrations of endog- cantly higher 2 weeks after hypophysectomy. enous cholesterol and cholesteryl esters were determined in separate experiments by quantitative TLC, using pure compounds as standards, and the TABLE 1. Effect of hypophysectomy (hypox) on cholesterol esterase activity of rat testes charring-photodensitometric technique (24, 25). The amount of endogenous cholesteryl esters in the Hydrolyzing Esterifying acAnimals" Testes wt (g) tivity'' activity'' homogenates was negligible compared to the amount of exogenous cholesteryl linoleate added to 3 Days posthypox (5) 2.6 ± 0.2'' 4.6 ± 0.7 2.5 ± 0.3 the incubation mixture. All incubations were car- Control 2.3 ± 0.1 Hypox (6) 4.4 ± 0.5 2.5 ± 0.3 ried out in duplicate, and the enzyme activities NS NS NS P value were expressed as nanomoles of cholesterol esteri- 7 Days posthypox Control (5) 2.8 ± 0.2 4.9 ± 0.9 2.3 ± 0.4 fied or cholesteryl ester hydrolyzed per h/mg proHypox (6) 1.8 ± 0.3 4.2 ± 0.6 1.3 ± 0.5 tein. NS P value
Vol. 103, No. 3 Printed in U.S.A.
Effect of Hypophysectomy of Cholesteryl Ester Synthesis and Hydrolysis in Testes and on Serum LecithinCholesterol Acyltransferase Activity in Rats* TAKEHIKO TAKATORIf AND 0. S. PRIVETT The Hormel Institute, University of Minnesota, Austin, Minnesota 55912 ABSTRACT. Studies are reported of the effect of hypophysectomy on cholesterol esterase activity of testicular tissue and serum lecithin-cholesterol acyltransferase (LC AT) activity in rats. The testes of male SpragueDawley rats of 200-255 g were excised from animals sacrificed at 3, 7, and 15 days after hypophysectomy. Assays for cholesterol-esterifying and hydrolytic activities of the testicular tissues of these animals, compared to control animals, showed that hypophysectomy decreased both cholesteryl ester synthesis and hydrolysis.
Hydrolytic activity was affected to a greater extent than esterifying activity. LCAT activity was significantly decreased by hypophysectomy compared to that of control animals. Although serum LCAT and testicular cholesterol esterase activities were decreased, the overall effect of hypophysectomy produced an increase in the level of serum cholesterol and cholesteryl esters. It is suggested that the role of essential fatty acids (EFA) in testicular
function is related to the utilization of cholesteryl esters in androgen synthesis. (Endocrinology 103: 748, 1978)
H
YPOPHYSECTOMY has a pronounced effect on the testes, producing large changes in the level and composition of the lipids (1-7), particularly cholesteryl esters (5-7). Among the changes in cholesteryl esters after hypophysectomy of mature rats is a large elevation of docosapentaenoic acid (7, 8), which is the major EFA in testes. In spite of the importance of EFA in testes development and spermatogenesis (9-15), the physiological role of these fatty acids in testicular function has not been elucidated. It has been shown that the mobilization of cholesteryl esters and the cholesterol fraction of these compounds are directly involved in androgen synthesis, which is under hormonal regulation of the pituitary (16-19). The studies of Behrman and Armstrong (20) and Behrman and Greep (21) showed that cholesterol esterase activity in ovarian tissue is increased upon LH treatReceived June 27, 1977. Address requests for reprints to: Dr. O. S. Privett, The Hormel Institute, University of Minnesota, 801 16th Avenue N.E., Austin, Minnesota 55912. * This work was supported in part by USPHS Grants AM-04942 from the NIH and HL-08214 from the Program Project Branch, Extramural Programs, the National Heart Institute, and the Hormel Foundation. f Present address: Department of Legal Medicine, Hokkaido University School of Medicine, Sapporo 060, Japan.
ment, suggesting that the activity of the enzyme system is a site of the regulation of steroidogenesis. Moreover, cholesterol esterase seemed to exhibit a selectivity for the hydrolysis of unsaturated cholesteryl esters (21). In a previous study (22), we investigated the enzymatic properties of cholesteryl ester synthesis and hydrolysis in rat testes. In the present study, the effect of hypophysectomy on enzyme activity in the synthesis and hydrolysis of cholesteryl esters in testes and on serum lecithin-cholesterol acyltransferase (LCAT) activity in rats is reported. Materials and Methods Animals Hypophysectomized and control male rats of the Sprague-Dawley strain, 200-225 g BW, were obtained from the Hormone Assay Laboratory (Chicago, IL), and fed Purina lab chow ad libitum. Groups of six hypophysectomized and five control animals were sacrificed at 3, 7, and 15 days after hypophysectomy. Enzyme preparation The animals were killed by decapitation and the testes were excised immediately. After removal of the capsule and visible blood vessels, each testis
748
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 19:26 For personal use only. No other uses without permission. . All rights reserved.
CHOLESTEROL ESTERIFICATION IN RAT SERUM AND TESTES was minced and then homogenized in 5 ml 0.25 M sucrose using a Potter-Elvehjem homogenizer, as previously described (22). Cell debris and nuclei were separated from the homogenates by centrifugation at 750 X g for 15 min, which provided a supernatant as the enzyme source. All procedures were carried out at 0 C and protein content of the enzyme preparations was determined by the Lowry method (23) using bovine serum albumin as a standard.
749
nanomoles of cholesterol esterified per min per liter of serum at 37 C by multiplying the percentage of labeled cholesterol esterified by the free cholesterol of the original sample. Significance of the data was determined by Student's t test.
Results
There was a large decrease in the size of testes of the hypophysectomized animals, as shown in Table 1. The effect of hypophysecSubstrates and other chemicals tomy on testicular cholesteryl ester synthesis [4-14C]Cholesterol (SA, 52.0 mCi/mmol) and [4- and hydrolysis are also shown in Table 1. M C]cholesteryl linoleate (SA, 20.9 mCi/mmol) were Seven days after hyophysectomy, cholesterylpurchased from New England Nuclear Corp. (Bos- esterifying activity was significantly decreased ton, MA). Impurities were separated from these compared to that of the control group. The preparations by thin layer chromatography (TLC) cholesteryl ester hydrolyzing activity started using plates coated with silica gel H and a solvent to show the effect of hypophysectomy at this system of petroleum ether-diethyl ether-acetic acid time and was significantly lower than that of (95:15:1, vol/vol). the control group 15 days after hypophysecBovine serum albumin (fraction V) was pur- tomy. In fact, hydrolyzing activity was inchased from Sigma Chemical Co. (St. Louis, MO), hibited to a greater extent than esterifying and cholesterol and cholesteryl linoleate were obactivity in the 15-day posthypophysectomized tained from the Lipids Preparation Laboratory of the Hormel Institute (Austin, MN). Scintillation group, inasmuch as the ratio of the activities chemicals were obtained from Packard Instrument was only approximately 1:1 compared to 2:1 in the controls. Co. (Downers Grove, IL). Hypophysectomy also caused a decrease in Cholesterol-esterifying and cholesteryl ester - LCAT activity within 7 days, as shown in hydrolyzing activities Table 2. Serum cholesterol was elevated 3 The incubation systems and analytical proce- days after hypophysectomy and remained dures were carried out, as previously described (22), high; the level of the cholesteryl esters inin which optimal conditions for esterification and creased more gradually and became signifihydrolysis were defined. Concentrations of endog- cantly higher 2 weeks after hypophysectomy. enous cholesterol and cholesteryl esters were determined in separate experiments by quantitative TLC, using pure compounds as standards, and the TABLE 1. Effect of hypophysectomy (hypox) on cholesterol esterase activity of rat testes charring-photodensitometric technique (24, 25). The amount of endogenous cholesteryl esters in the Hydrolyzing Esterifying acAnimals" Testes wt (g) tivity'' activity'' homogenates was negligible compared to the amount of exogenous cholesteryl linoleate added to 3 Days posthypox (5) 2.6 ± 0.2'' 4.6 ± 0.7 2.5 ± 0.3 the incubation mixture. All incubations were car- Control 2.3 ± 0.1 Hypox (6) 4.4 ± 0.5 2.5 ± 0.3 ried out in duplicate, and the enzyme activities NS NS NS P value were expressed as nanomoles of cholesterol esteri- 7 Days posthypox Control (5) 2.8 ± 0.2 4.9 ± 0.9 2.3 ± 0.4 fied or cholesteryl ester hydrolyzed per h/mg proHypox (6) 1.8 ± 0.3 4.2 ± 0.6 1.3 ± 0.5 tein. NS P value
