72—4446

CHENG, Hsien Chen, 1927- ISOENZYMES OF IN RAT TESTES AT VARIOUS DEVELOPMENTAL AND ENDOCRINE STATES.

The Ohio State University, Ph.D., 1971 Physiology

University Microfilms, A XEROX Company , Ann Arbor, Michigan

THIS DISSERTATION HAS BEEN MICR0FLIMED EXACTLY AS RECEIVED i s o e n z y m e s o f h e x o k i n a s e in r a t t e s t e s a t v a r i o u s

DEVELOPMENTAL AND ENDOCRINE STATES

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

%

Hsien Chen Cheng, B.S., M.S.

The Ohio State University 1971

Approved by

Department of Dairy Science PLEASE NOTE:

Some Pages have Indistinct print. Filmed as received.

UNIVERSITY MICROFILMS ACKNOWLEDGMENTS

I would like to thank Dr. N. L. VanDemark, my Department

Chairman, for providing me with financial support as well as labora­ tory facilities to conduct my research work. I would like to thank especially my advisor, Dr. W. R. Gomes, for his invaluable and ingenious guidance. I am also grateful for his patience and genero­ sity in sparing his precious time.

I would like to give my special thanks to Mrs. Claudia Jenkins for her artistic talents in the preparation of the graphs herein contained.

To my wife, Mrs. Jane H. G. Cheng and son Christopher Cheng,

I am grateful for their forever patient and constant encouragement.

ii VITA

September 21, 1927 .... B o m - Kiang-Su, China

1956 ...... B.S, National Taiwan University, Taipei, Taiwan, China

1956-1959...... Teaching Assistant, National Defense Medical School, National Taiwan University, China

1962 ...... M.S. Washington University, St. Louis, Missouri

1962-1963...... Electron Microscopy Training Program, Department of Anatomy, Washington University, St. Louis, Missouri

196^-1968...... Technical Assistant, College of Medicine, The Ohio State University, Columbus, Ohio

1968-1971...... Graduate Research Associate, Department of Dairy Science and Animal Reproduction Teaching and Research Center, The Ohio State University, Columbus, Ohio

PUBLICATIONS

Cheng, H. C, Effect of Cortisone on Chick Embryo Development. B.S. Thesis. National Taiwan University, 1956.

Cheng, H. C. Effect of Blindness on Alkaline Phosphatase in Mouse Intestinal Epithelium. M.S. Thesis. Washington University, St. Louis, Mo. 1962.

Couri, D., H. C. Cheng, and C. A. Angerer. Pyridine Nucleotides Content in Human Placenta. Fed, Proc. 25:642, 1966.

FIELDS OF STUD!

Major Field: Physiology of Animal Reproduction

Studies in Physiology of Reproduction. Professor N. L. VanDemark Studies in Endocrinology of Reproduction. Professor W. R. Gomes Studies in Biochemistry. Professor G. S. Serif Studies in Pharmacology and Drug . Professor D. Couri

ill TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS...... ii

VITA...... iii

LIST OF TABLES...... v

LIST OF FIGURES...... vi

PREFACE...... viii

INTRODUCTION...... 1

LITERATURE REVIEW ...... 3

Introduction Properties of Distribution and Functions of Hexokinases Adaptation of and Hexokinase Hormonal Control of Hexokinase Activity

MATERIALS AND METHODS ...... 15

Experimental Animals Histological Procedures Extraction Chromatographic Procedures Enzyme Assay Electrophore sis

RESULTS ...... 19

Body Weight, Testis Weight and Testis Histology Column Chromatography of Hexokinase Electrophoresis of Hexokinase Isoenzymes

DISCUSSION...... 44

SUMMARY ...... 48

REFERENCES...... 49

iv LIST OF TABLES

Table Page

1. Body and Testis Weights of Experimental Rats , . 20 2. Sunmary of Hexokinase Isoenzymes Identified by DEAE Cellulose Chromatography...... 39

v LIST OF FIGURES

Figure Page

1. Photomicrographs of normal rat testes...... 22

2. Photomicrographs of testes from normal and HCG- treated rats ...... 24

3. Photomicrographs of testes from hypophysectomized, hypophysectomized-HCG-treated, and cryptorchid rats. 26

4* Chromatographic separation of brain hexokinase iso- 30 from 20-day-old rats ......

5. Chromatographic separation of brain hexokinase iso­ enzymes from adult rats...... 30

6 . Chromatographic profiles of hexokinase isoenzymes in testes from 20-day-old r a t s ...... 32

7. Chromatographic profiles of hexokinase isoenzymes in testes from 30-day-old rats ...... 32

8 . Chromatographic profiles of hexokinase Isoenzymes in testes from 40-day-old r a t s ...... 33

9. Chromatographic profiles of hexokinase isoenzymes in testes from adult rats...... 33

10. Chromatographic separation of hexokinase isoenzymes of testis tissue from 20-day-old rats treated for 10 days with HCG (100 IU/dsy; Ayerst Lab. Inc.). . . 36

i 11. Chromatographic separation of hexokinase isoenzymes of testis tissue from 20-day-old rats treated for 10 days with HCG (100 IU/day; Sigma Chemical Co.). . 36

12. Chromatographic separation of hexokinase isoenzymes of testes from rats hypophysectomized 3 weeks. . . . 37

13. Chromatographic separation of hexokinase isoenzymes of testes from hypophysectomized rats treated for 7 days with HCG...... 37

14. Chromatographic separation of hexokinase isoenzymes of testes from hypophysectomized rats treated for 14 days with H C G ...... 38

vi Figure Page

15. Chromatographic separation of hexokinase iso­ enzymes of testes from rats cryptorchid for U weeks ...... 38

16. Hexokinase isoenzymes separated hy polyacryla­ mide disc gel electrophoresis of experimental tissues ...... 42

vii PREFACE

Significance of this Research

In the present age, maintenance, improvement or reversible inhibi­ tion of normal reproduction in man and animals may become vital to the world*s economy or its very existence. A number of studies have been conducted to determine the efficacy of several specific treatments an testis function, but it appears that successful regulation of the organ will not be accomplished until basic controlling mechanisms within it are better understood.

The testis is known to be very sensitive to a number of influ­ ences, including environmental, endocrine, and biochemical changes.

Many of these treatments result in altered energy metabolism, suggest­ ing that pathways of glucose metabolism might be altered. Since the first step in glucose utilization, biochemically, involves the enzyme hexokinase, this study was undertaken to evaluate the nature and con­ trolling factors for testicular forms of this enzyme.

viii INTRODUCTION

It has been shown that normal adult testes of men (Blanco and

Zinkham, 1963), rabbits, mice, dogs, guinea pigs, bulls and pigeons

(Zinkham, Blanco and Clowry, 1964) contain an unusual isoenzyme of lactate

dehydrogenase. This isoenzyme has been designated LDH-X; it is composed

of subunit C, which is different from subunits of A and B, present in

other LDH isoenzymes (Appella and Markert, 1961). The developmental

changes in LDH isoenzymes of the testis have been studied by Goldberg and

Hawtrey (1967), who demonstrated mouse testis LDH-X appears between 15

and 17 days after birth, which corresponds to the first appearance of

pachytene primary spermatocytes (Nebel, Amorose and Haekett, 1961).

Blackshaw and Elkington (1970) demonstrated that LDH-X isoenzymes

appear between 20 and 30 days of age in the rat and are also associated

with the seminiferous tubular epithelium.

Studies utilizing DEAE-cellulose chromatography of rat liver (Gonzalez

et al.. 1964) and starch gel electrophoresis of rat epididymal fat pad

(Moore §X Al*» 1964) have revealed that mammalian hexokinase consists of

a variable number of isoenzymes. Recently, Katzen (1967) demonstrated an

isoenzyme of hexokinase in mature rat testes, which he called "sperm type".

Pilkis, Hansen and Krahl (1968), using starch gel electrophoresis, demon­

strated a testicular isoenzyme of hexokinase in gerbils and hamsters.

A testis specific type of hexokinase, Hex-t, has also been reported in

Drosophila melanogaster at the early pupal stage (Murray and Ball, 1967).

These workers demonstrated that Hex-t is present in the testes of females carrying transformer gene (tra/tra); however, testes obtained from this type of female showed no mature spermatozoa histologically. The original purpose of this investigation was to determine whether

/ the so-called "sperm type" isoenzyme of kexoklnase in rat testis is really from the spermatozoa, from other cellular components of the testis, or due to other factors which might influence the first appearance of this specific isoenzyme. This question was raised on the basis of the report by Katzen al. (1968) that this isoenzyme was present not only in the adult testis, but also in the epididymal fat pad, suggesting that the term "sperm type" was a misnomer. If this specific type of hexokinase appears only in the adult male rat, then its appearance may be regulated by the presence of the male hormone. Singhal and Ling (1969) have shown that testosterone can induce hexokinase synthesis in rat seminal vesicle.

A second question is which cell type in the testis contains this specific type of hexokinase isoenzyme. The testis is composed of several types of cells; the two major types are the Leydig cells and the germinal cells. Whether this hexokinase Isoenzyme is synthesized by either of these cells, synthesized by both, or present in neither, is not known.

The present study should also help our understanding of interrelation­ ships between the two major cellular components of the testis. The hor­ mones secreted by Leydig cells are known to influence the maturation and metabolism of the germinal epithelium, but the site of such effects is unknown. In the present consideration, influences of testosterone on testicular hexokinase are of interest.

The research described in this thesis was undertaken to determine developmental changes and hormonal influences on rat testis hexokinase isoenzymes. It is hoped that the data will serve as a foundation for future research into energy metabolism in the testis at the enzyme level. LITERATURE REVIEW

Introduction

In rat testis, glucose is an essential substrate for the mainte­

nance of tissue integrity (Mancine e± sX., I960). The utilization of

glucose by any tissue first involves the phosphorylation of glucose.

The enzyme which participates in the phosphorylation of glucose or other

hexoses is hexokinase. In considering the part played by the hexokinase

in the overall control of hexose utilization in a tissue, we have to

consider the following properties; (l) Their location within the cell,

for example, particulate fraction and/or soluble fraction, (2 ) their maximal activity and the possibility of this being achieved in vivo.

(3) their specificity, (4) their affinity for substrates and (5) any other controlling factors, such as hormonal influences on certain specific tissues.

It has been recognized that multiple forms of many enzymes may exist within a single organism. This is very important not only for the study of enzymology itself, but also for the study of genetic con­ trol and metabolic regulation. There are many instances of tissues containing multiple forms of enzymes, such as carbonic anhydrases

(Edsail, 1968), creatine (Dawson et al., 1968), phosphofructo- kinases (Layzer and Conway, 1970), aldolases (Rutter et al.. 1968), glucose-6-phosphate dehydrogenases (Kirkman and Hanna, 1968; Shaw and

Koen, 1968), and lactic dehydrogenases (Kaplan, 1968). Recently, by a variety of chromatographic and electrophoretic techniques, Gonzalez et al. (1964) and Katzen and Sc hi rake (196?) demonstrated that hexo- kinases can be resolved into four molecular forms, or isoenzymes in liver homogenates. Gonzalez e£ al. (1964) named these isoenzymes A,

B, C and D, according to the increasing degree of retention on DEAE- cellulose columns; however, Katzen and Schimke (1965) referred to them as I, II, III and IV, based on their increasing mobility during electro­ phoresis. Each peak of activity eluted from the DEAE-cellulose column was found to correspond to an electrophoretically different hexokinase type, i.e. A was the same as I, B equals II, etc. Hexokinase type IV was found to be identical to the glucokinase enzyme reported by Walker

(1963). This type IV (glucokinase) has a high K,q value toward glucose, as compared with the other three isoenzymes of hexokinase.

Properties of Hexokinases

The hexokinases found in most animal tissues are capable of phos- phorylating a number of different acceptor molecules. Some compounds are not phosphorylated, but an affinity is quoted because they act as competitive inhibitors at the substrate-. A valuable method for correlating the maximum rate, V ^ y , and was devised by Sols and

Crane (1954) as follows:

VM Y (substrate) K_ (glucose) Phosphorylation coefficient = --- — ------— X ----- ;----- (substrate) ^max (glucose

Glucose is used as the reference substrate because it has the highest phosphorylation coefficient and is also the physiological substrate.

The phosphorylation coefficient then gives a good indication of the

'physiological suitability' of any other substrate for the hexokinase.

Hexokinases can utilize analogues of ATP, for example ITP, as the phosphoryl donor only at extremely low rates. Of greater interest is the problem of the true nature of the phosphoryl donor at the active

site of the enzyme and the association of Mg++ ions with the donor.

As with many of the other , optimum activity is obtained when the ratio of ATP molecules to Mg++ ions is about unity.

Using partially purified brain hexokinase, Crane and Sols (1954) confirmed an earlier finding that inhibition by glucose-6-phosphate was non-competitive. The Kj for glucose-6 -phosphate, 4xlO-i*M, and the specificity of this inhibition are quite different from those of the substrate, which seems to inhibit only type III hexokinase.

The binding of the donor nucleoside triphosphate, carrying a net negative charge, may involve a positively charged group on the enzyme surface. The hexokinases depend for their activity upon the presence of free sulfhydryl groups. This has been particularly noted for ketohexo- and glucokinase (Salas e£ a^., 1965). The inhibitory effects of compounds such as p-chloromercuribenzoate and p-chloromercuriphenyl- sulfonate are reversed by addition of a thiol reagent such as cysteine

(Ponz and Llinas, 1963). At least two of the enzymes also have a very definite requirement for K+ or other univalent cations. For ketohexo- kinase a for K+ ions of 5.2 mM has been quoted. K+ and Rb+ ions are the most active, with NH^+, Na+, Cs+ and Li+ decreasingly effective in this order (Parks al-» 1957). K+ ions also protect glucokinase against inactivation. Presumably the single charge and ionic size of

K+ exert a modifying influence on the electron-density distribution.

Distribution and Functions of Hexokinases

The relative distribution of enzyme activity among the four Isoenzymes appears specific for each tissue and species. Not all

mammalian tissues have the full complement of the four isoenzymes; each

tissue may contain two or more. Hexokinase isoenzymes of a number of different cells and tissues are discussed below.

Ervthrocvte. Hexokinase Isoenzymes have been demonstrated in erythrocytes of many different species (Malone al., 1968; Kaplan,

1968). Kaplan (1968) found that human erythrocyte hexokinase type I was split into two, viz. band type IA and Ip by electrophoresis. The

■type Ip is more intense in umbilical cord blood. No type II enzyme was found in umbilical cord blood. The double type I band was also present in adult rabbit red blood cells.

Tumor cells. A good deal of work has been done on hexokinases in many different types of tumors in the hope that basic differences in the specificity of tumor hexokinase from that of the corresponding normal cell would be revealed, providing a means of destroying malignant cells by therapeutically inhibiting the tumor enzyme.

MeComb and Yushok (1959) and Saito and Sato (1971), in their studies of particulate bound hexokinase in rat ascites hepatoma cells, demonstrated that mitochondrial bound hexokinase contained more type I than type II isoenzyme; however, it is generally accepted that particu­ late-bound hexokinase in malignant cells is mainly type II enzyme and that type II also predominates in the soluble fraction (Kosow and Rose,

1968; Gumas and Green&lade, 1968). These divergent results may be due to the different pH of the buffers used for extraction of the enzymes;

Borrebaek (1970) demonstrated that the percentage of mitochondrial- bound hexokinase varies at different pH. Manjeshwar e£ (1965) in a study of hepatocarcinogenesls demon­

strated that hexokinase levels were elevated during the early periods

of regeneration after hepa tec tony and return to normal levels in one or

two weeks. However, glucokinase level was low during the early period after hepatectony, but reached normal in 4 to 7 days. After rats were treated with dimethylsminoazobenzene (DAB) or 3-methyl DAB, known carcinogens, glucokinase was absent but hexokinase levels were moderate to high in cholangiocarcinomas. Both glucokinase and hexokinases were present in hepatocellular carcinomas.

Brain. Brain tissue is very complex. Its dependence upon glucose as an energy source is well established. Of all the glycolytic enzymes, perhaps only hexokinase and are associated with mitochondria. Only two types of hexokinase isoenzymes are present in the brain. Type I has a very high activity, and type II exhibits very weak activity (Crossbard and Schimke, 1966). Recently, Rose and Warms

(1967) demonstrated that mitochondrial hexokinase can be released and rebound by the presence or absence of glucose-6-phosphate. On the other hand, Thompson and Bachelard (1970) found that mitochondrial and cyto­ plasmic hexokinases are basically similar in properties. Wilson (1967) suggested that latent mitochondrial hexokinase I may play an important part in the physiological function of the enzyme. Chance and Hess

(1956) also have suggested that ADP formed by mitochondrial-bound hexo­ kinase in some ascites tumor cells readily diffuses into the mitochondria and stimulates their respiration.

Muscle. Electrophoretic techniques have shown that muscle hexokinase consists of two types, type X and type II, in many different

species (Grossbard ei si., 1966). In rats, mice, hamsters and cattle,

type II far exceeds (quantity and activity) type I. On the other hand,

guinea pig, rabbit and monkey skeletal muscle contain approximately

equal amounts of the two Isoenzymes. Recent investigation by Ilyin,

Pleskov and Razumovskaya (1970) demonstrated that the ratio between iso­

enzymes I and II in rabbit skeletal muscle is about equal in normal

animals. However, the ratio changes to 1:5 after denervation. This

change is due both to a decrease in hexokinase I and to a very large

increase of hexokinase II. In muscle of the embryo, the ratio is about

the same as denervated adult muscle. Hernandez and Crane (1966) have

found that in heart muscle there is an equilibrium between the soluble

and particulate-bound forms of hexokinase which depends upon pH and

ionic strength. Glucose-6-phosphate, probably by acting at the specific

regulatory site, Induces solubilization of the heart hexokinase. The

significance In vfrvo of these observations is not known.

Adipose tissue. In a study of white adipose tissue, DiPietro

(1963) suggested that a single enzyme catalyzes the phosphorylation of

glucose and . However, Moore ei al. (1964), using a starch gel electrophoretic technique, found two types of isoenzyme in adipose

tissue. When animals were starved for 48 hours, the high Kg, enzyme was

reduced to a very low level but returns to normal values during a 24-hour

refeeding period. Whether this "glucokinase" in adipose tissue is the same as the glucokinase in liver is not known. Katzen and Schimke

(1965) demonstrated that type I and type II hexokinase isoenzymes in adipose tissue are age-dependent. In young rats, largely type II was present; however, in old rats, the reverse was true. Grossbard and

Schimke (1966) separated the adipose tissue hexokinase isoenzymes on

DEAE cellulose. They used Osbome-Mendel albino rats, weighing 150-

175 grains, and found that type II activity was much higher than type I.

Recently, Borrebaek (1970) found that a large portion of the adipose tissue hexokinase activity was associated with the mitochondria of carbohydrate-fed rats as compared with the fasted rats. Increased mito­ chondrial bound hexokinase was observed in fat pads incubated jjj vitro in the presence of glucose. These workers also found that the amount of epididymal adipose tissue hexokinase associated with the mitochondria varied with pH, due to different stabilities of the binding. Highest stability was observed when using a homogenizing buffer adjusted to pH 8.0 to 8.5. The binding of hexokinase to mitochondria was stabilized by glucose In vitro. The percentage of the adipose tissue hexokinase associated with the mitochondrial fraction was increased in carbohydrate- fed rats as compared with fasted rats. This increase was specifically related to hexokinase type II. Pilkls (1970), in a study of hormonal control of hexokinase in animal tissues, found that adipose tissue hexo­ kinase in hypophysectomized rats was about one-half of the normal rats.

In thyroidectomlzed rats, adipose tissue hexokinase was also reduced to about one-half normal, but this reduction could be restored by injection of thyroxine.

Liver. In monogastric animals, digestible carbohydrates of the food are absorbed as monosaccharides and transported to the liver. 10

There they may be stored by conversion to glycogen, or they may be

taken to adipose tissue for storage in the form of fat.

One important aspect of hexose utilization by the liver is that

the liver cell membrane appears to be freely permeable to hexoses, per­

mitting rapid equilibration of extracellular and intracellular concen­

tration (Cahill a 1.» 1959).

Recent studies have demonstrated the presence of more than one

hexokinase of low Km in liver tissue. Three liver hexokinases plus

glucokinase have been separated by HEAR cellulose chromatography

(Gonzalez jjl., 1964; Vinuela £± Al-, 1963; Grossbard and Schimke,

1966; Katzen and Schimke, 1965). Multiple forms have also been separated by starch gel electrophoresis of extracts both from rat liver and human cell culture (Katzen £l Al., 1965; Katzen and Schimke, 1965; Katzen,

1967). In a developmental study, Walker (1965) found that glucokinase is absent from fetal liver and develops immediately after birth in the guinea pig, but first appears about 16 days after birth in the rat and reaches adult levels at about 26 to 28 days of age. also appears to develop after birth.

The molecular weight of hepatic glucokinase, as determined by

Pilkis, Hansen and Krahl (1968), is about 48,000, or one-half of the low Km hexokinases. Hepatic fructokinase also have been purified and studied by Sanchez (1971). has not been studied exten­ sively. is metabolized faster by liver tissue from new born rats than by adult liver tissue (Segal e± Al-» 1963), due to higher galactokinase activities during the newborn period (Cuatrecasas and Segal, 1965). 11

Testis. Hensen ei al. (1967) demonstrated that liver hexokinase

IV (glucokinase), like hexokinase II of adipose tissue, has electro-

phoretically "fast" and "slow" forms. The fast form, which migrates

like glucokinase when electrophoresed, disappeared during fasting and

in diabetes and was restored by refeeding and insulin administration,

respectively. In the testis, glucokinase is present only as a slow

migrating form, which has a high toward glucose and a lower molecular weight (determined on Sephadex G-100) than the low hexokinase.

Katzen (1967), using starch gel electrophoresis, demonstrated a

slow moving band of hexokinase in rat testes; this particular band was not present in prepubertal testes, but was found in adult testis. Since

the isoenzyme appeared to be due to the appearance of spermatozoa,

Katzen (1967) designated this particular band of hexokinase “sperm type".

He also demonstrated that this "sperm type" hexokinase is present in the epidldymal adipose tissue. Filkis, Hansen and Krahl (1968) also found an extremely slow moving band of hexokinase in testis extracts of gerbils and hamsters, and Murray and Gall (1967) demonstrated a very slowly moving band of hexokinase, Hex-t, in testes from Drosophila melanogaster. It appears at the early pupa stage and is also present in testes of females carrying transformer (tra/tra) gene.

Adaptation of Glucokinase and Hexokinase

One of the important ways in which a change of enzyme activity can occur is by an alteration in the enzyme content of the cell. The unique high Kjq glucokinase is an adaptive enzyme. The total glucose pbosphorylating activity falls by 50 percent or more after 48 hours 12

starvation, and this is due to the fall of glucokinase but not hexo­

kinase activity (Walker, 1965; Pilkis, 1970). The glucokinase activity

falls to a very low level in alloxan diabetic rats. When the starved nondiabetic rat is refed, glucokinase activity is restored to normal

in few hours. When alloxan diabetic rats were treated with insulin, glucokinase activity was also restored in 24 hours (Vinuela ei al.,

1963; Walker, 1965; Salas ei al., 1963; Sharma et al., 1963, 1964; Sols

£± fll., 1964; Hansen et al.. 1967a, 1967b; Pilkis, 1970). The increase of glucokinase activity is apparently due to de novo synthesis of enzyme

(Pilkis, 1970).

Adipose tissue hexokinase II also appears to be an adaptive enzyme

(Moore et al. r 1964; Katzen and Schimke, 1965; Hansen £i Al-, 1967a,

1967b, 1970; Borrebaek, 1967, 1970; Pilkis, 1970). The level of hexo­ kinase II decreases significantly after 43-72 hours of fasting and is restored 10-24 hours after refeeding. Restoration can be blocked by cycloheximide or actinonyein D administration.

Hormonal Control of Hexokinase Activity

Salas et al. (1963), Sols et al. (1964) demonstrated that gluco­ kinase in rat liver disappears in diabetic animals andreappears within a few hours after insulin administration. Glucagon and epinephrine administration do not affect glucokinase activity, suggesting that insulin directly or indirectly induces glucokinase. In adult rabbits,

Ilyin (1964) demonstrated that cortisone acetate inhibited both mito­ chondrial bound and soluble fraction Of cytoplasmic hexokinases; how­ ever, embryonic rabbit liver did not response to cortisone acetate 13 injection. Machiya and Hospya (1969) demonstrated that the activity of liver and muscle type II hexokinase in diabetic rats and type IV in diabetic liver were markedly reduced, but returned following insulin injection. Following hypophysectoay, liver glucokinase (Sharraa ei al.T

1964; Pilkis, 1970) and total adipose tissue hexokinase (Pilkis, 1970) were subnormal. Chronic injection of thyroxine to hypophysectomized rats resulted in almost no increase in glucokinase activity and a very large increase in low hexokinase activity. On the other hand, chronic injection of hydrocortisone to hypophysectomized rats greatly increased glucokinase activity, but hexokinase activity remained low.

In thyroidectomized rats, liver glucokinase was normal, but adipose tissue hexokinase II was decreased by half. Injection of thyroxine restored the type II enzyme. Baquer and McLean (1969) showed that 4 hours after injection of estradiol, no change occurred in uterine hexokinase in rats, but there was a change in the distribution between the particulate and soluble fractions. Hexokinase type I binding to mitochondria increased 88 percent. No change was found in binding of type II hexokinase. Significant increases in the total hexokinase of the uterus were found 12 hours after injection of estradiol. At 96 hours after injection, hexokinase type I increased threefold in the soluble fraction and 13 times in the mitochondrial fraction. Hexokinase type II increased 10 times in both fractions.

Katzen (1967), in a study of hexokinase type II isoenzyme in epididymal fat pad, cardiac muscle and skeletal muscle, suggested that action of insulin is to stabilize type II hexokinase isoenzyme through a thioldlsulfide interexchange between the hormone and the enzyme.

However, Murakami and Ishibashi (1970), using granuloma cells, sug­ gested that Insulin, a receptor protein and type II hexokinase may form a complex which is sensitive to sulfhydryl compounds; the sug­ gested mechanism of insulin action is to Induce synthesis of this receptor protein. MATERIALS AND METHODS

Experimental Animals

All animals used in this study were Wistar strain rats purchased from a commercial supplier or raised in the animal colony of the Animal

Reproduction Teaching and Research Center, The Ohio State University.

For the study involving developmental changes in hexokinase enzymes, rats 20, 30, 40, and 60 days of age and normal adult rats weighing 300-

400 g were used.

For studies involving gonadotrophin treatment, both immature and hypophysectomized rats were used. In the first case, rats 10 days old were treated with 50 TU of HCG s.c. twice daily for 10 days and sacri­ ficed at 20 days of age. Hypophysectomized rats weighing 150-200 g were obtained from the Charles Rivers Breeding Laboratories. Three weeks after hypophysectoray, HOG injections were begun (100 IU/rat/day i.p.) and continued for 7 or 14 days. Rats were sacrificed the day after the final injection and the testes were recovered.

In order to detennine the effects of abdominal temperatures on hexokinase isoenzymes, artificial cryptorchidism was produced in mature rats as described by Free ej al. (1969). Four weeks later, the rate were sacrificed and the testes recovered.

Except for hypophysectomized rats, all animals were maintained on a regular diet of Purina laboratory chow with free access to fresh water. Hypophysectomized rats were maintained on regular Purina labora­ tory chow supplemented with 0.9% NaCl. 15 16 Histological Procedures

Samples of testes of each experimental group were fixed in Bouin's solution, embedded in paraffin, sectioned at a thickness of 5 u, and stained with hematoxylin and eosin.

Enzyme Extraction

After testes were surgically removed from anesthetized rats, the tunica albuginea and testicular blood vessels were quickly removed and the testes were put into 5.0 ml. of Tris-Cl buffer (pH 7.4, containing

-5mM EDTA-Nag* 5mM 2-mercaptoethanol and lOmM glucose). Testis tissues were pooled until a 5.0 ml. sample was collected. It was then homogenized in a glass homogenizer fitted with teflon pestle and centrifuged at approximately 45,000 x g for 60 min. in Sorvall-RC2-B refrigerated cen­ trifuge. The supernatant was collected and the volume of the supernatant was measured. The crude extracts were either used immediately or stored at -20° C.

Chromatographic Procedures

Dry DEAE-cellulose was washed with 500 ml. of distilled water, and the sluny was resuspended in 500 ml. 1.0 N NaOH. After centrifugation, the slurry was recovered and rewashed with 1.0 N NaOH until no more color was removed. The slurry was then washed with 1.0 N RC1, filtered and washed with distilled water until the pH of the wash was the same as the distilled water. The cellulose was rewashed with 1.0 N NaOH, centrifuged, and washed with distilled water until no remaining alkali was present in the cellulose. Finally, the washed DEAE cellulose was suspended in 0.01 M potassium phosphate buffer, pH 7.0, for equilibra­ tion.

i 17

A glass column 0.9 x 30 cm. (l.d. x height) was poured with the

suspended DEAE cellulose and with 100 ml. of 0.01 M phosphate "buffer.

The column was then moved to a room maintained at 3° C. and washed with

100 ml. of 0.01 M phosphate "buffer containing 3ntM EDTA-Na2 » 5mM 2-

mercaptoethanol and lOnM glucose. The pH of the buffer passed through

the column was checked for assurance of equilibration.

Enzyme Assay

Hexokinase activity was assayed spectrophotometrically at 37° C.

by measuring glucose-6-phosphate formed. The routine assay mixture con­

tained 74 mM Tris-Cl (pH 7.4), 25 mM glucose, 0.3 nM TPN+, 3.7 nM ATP,

7.4 mM MgClg, 5.0 mM 2-roercaptoethanol, and 0.9 international units of

glucose-6-phosphate dehydrogenase in a 3-ml. system. Continuous record­

ings of the increase in optical density at 340 mu resulting from TPN+

reduction were made using a Beckman D3 spectrophotometer coupled with a

Sargent Model SR recorder. Reaction was initiated by the addition of

the enzyme. One unit of hexokinase activity was defined as the amount

of enzyme which transformed 1 umole of substrate per min. at 37° C. in the assay system used.

Electrophoresis

A standard reagent kit was purchased from the Canaleo Company,

Rockville, Md. This reagent kit contained a separating gel of 1% acrylamide, which covers a protein range of 10,000 to 1,000,000 molecu­ lar weight, with maximum resolution from 30,000 to 300,000 m.w. Tris- glycine buffer, adjusted to pH 8.5 and containing 5 mM EDTA-Nag and

5 mM 2-mercaptoethanol was used. Electrophoresis was carried out at 18

4-5° C for 3-4 hour8 with a potential gradient of 2.5 - 3.5 mArap per tube. Gels were stained for hexokinase activity at room temperature in the absence of light by immersion in 0.01 M Tris-Cl developer solu­ tion at pH 7.4. The developer contained 0.3 nM TPN+, 5.0 mM MgC^,

5.0 nM ATP, 0.4 international units per ml. of glucose-6-phosphate dehydrogenase, 40 ug per ml. of phenazine methosulfate, 400 ug per ml. of nitro blue tetrazoliuin and 0.25 M glucose. The gel was destained In

1% acetic acid.

Enzymes were prepared for electrophoresis by concentrating crude extracts or column eluates by pervaporation through dialysis tubing.

Concentrated samples were mixed with 40£ sucrose (1:1) and applied above the previously polymerized separating gel. RESULTS

Body Weight. Testis Weight and Testis Histology

During maturation in the rat, body and testis weights increased

steadily with time (Table 1). Associated with body growth and increas­

ing testis weight, seminiferous tubule diameter increased. The first

appearance of pachytene primary spermatocytes occurs at about 30 days

of age and spermatozoa appear in testes of the 4.0-day old rat (Clermont

and Perey, 1957). Testis weights of HCG treated 20-day old rate were

heavier than normal 20-day old rats (P<0.01). Average testis weight

of the hypophysectomized and cryptorchid rats found in this study are

similar to those reported by others (Free et al., 1969; Massie, 1970)

and are smaller (P<0.01) than testes from normal rats of similar age

and weight. Treatment of hypophysectomized rats with HCG for 7 or 14

days resulted in an apparent, but not significant, increase in testis weight (Table 1).

Histology

As shown in fig. 1, morphology of the rat testis reflects the growth and functional changes that occur during development from the prepubertal testis (fig. la) to the normal adult testis (fig. Id).

These changes have been previously described in detail (Clermont and

Perey, 1957), The testes from HCG-treated immature rats have been divided into two groups, as shown in fig. 2. When 10-day old rats were treated for 10 days with HCG purchased from Ayerst Laboratories

(fig. 2b) testes exhibited marked proliferation of interstitial materials, and disrupted, denuded tubules, as compared to testes from normal 20-day

19 20 TABLE 1

BOOT AND TESTIS WEIGHTS OF

EXPERIMENTAL RATS

Number Body wt. Testis Weight Group® of rats (e) g/pair g/100 g body wt.

Normal Rats

20 days old . 104 36.4 0.17 0.44 30 days old 4? 71.9 0.52 0.72 40 days old 25 143.9 1.17 0.87 60 days old 5 279.0 2.60 0.93 . Adult 6 499.4 3.33 0.66 HCG-treated Ratsb

Ayerst - HCG 36 46.4 0.40 0.83 Sigma - HCG 13 46.6 0.42 0.90 Hypophysectomized Rats°

HypoX control 10 149.9 0.53 0.33 HCG - 7 days 8 146.4 0.63 0.43 HCG - 14 days 8 171.4 0.71 0.41

4-week Cryptorchid Rats 11 503.5 0.77 0.15

a See text for details of treatments b 20 days old c Adult rats 21

FIGURE 1

Photomicrographs of normal rat testes (all x 200).

a. 20-day-old rat

b. 30-day-old rat

c . 40-day-old rat

d . adult rat

FIGUHE 2

Photomicrographs of testes from normal and HCG-treated rats (all x 200).

a. 20-day-old normal rat

b. 20-day-old rat treated for 10 days with

HCG (100 IU/dey; Ayerst Lab. Inc.)

c. 20-day-old rat treated for 10 days with

HCG (100 IU/day; Sigma Chemical Co.) m FIGURE 3

Photomicrographs of testes from hypophysectomized, hypophy sectomized HCG-treated, and cryptorchid rate (all x 200).

a. Normal adult rat

b. Rat 3 weeks after hypophysectomy

c. Hypophysectomized rat treated for 7 days with HCG

(100 IU/day; Sigma Chemical Co.)

d. Hypophysectomized rat treated for 14 days with HCG

(100 IU/day; Sigma Chemical Co.)

e. Cryptorchid rat 4 weeks after translocation of

testes into the abdomen

27 old rats (fig, 2a). On the other hand, treatment of similar rats with the same dose of HOG purchased from Sigma Chemical Company (fig. 2c) resulted in similar proliferation of interstitial elements, but tubular morphology appeared unchanged from normal. The reasons for the differ­ ing responses to the two hormone preparations are unknown.

Three weeks after hypophyeectomy, tubular diameter within testes was markedly reduced (fig. 3b), Leydlg cells were reduced in size and number and mature spermatozoa were absent from the testes. Treatment of hypophysectomized rats with HCG (Sigma) for 7 (fig. 3c) or 14 days

(Fig. 3d) resulted in no apparent change in tubular morphology; the one- week treatment was also without observable effect on interstitial mate­ rials, but the 14-day treatment dramatically increased these elements.

Artificial cryptorchidism for 4 weeks resulted in almost complete degeneration of seminiferous tubules (fig. 3e), with only Sertoli cells and basal spermatogonia remaining. Interstitial materials are present in normal, or increased, amounts and appear histologically normal.

Column Chromatography of Hexokinase

Hexokinases in rat testes have previously been separated by starch gel electrophoresis (Katzen, 1967). Katzen designated the isoenzymes types I, II, III and IV in order of increasing mobility during starch gel electrophoresis. In the present study, brain tissue was used to identify the type I hexokinase. The basic principle of the elution of various hexokinase types was in the increasing molarity of salt

(potassium chloride) in 0.01 M potassium phosphate buffer at which the different types were eluted from the DEAE cellulose column. Thus, the order of their elution frcm DEAE cellulose column was in the order of 28

type I to type IV with increasing KC1 concentration.

Brains from 20-day old and adult rats were quickly removed, washed

in trie-Cl buffer 3 times to remove blood contamination, and homoge­

nized in a Waring blendor. The homogenate was centrifuged in a Servall

refrigerated centrifuge at 45,000 x g for 60 min. and the supernatant

was put on a prepared column. The column was then washed with 30 ml.

of phosphate buffer (pH 7), which was equivalent to about 1.5 hold back

volumes. The column was then developed by continuous gradient elution

from 0 to 0.6 M of KC1. Figures 4 and 5 illustrate the hexokinase iso­

enzymes eluted from brain extracts of 20-day old and normal adult rats

respectively. Type I activity is much higher than type II as reported

by Grossbard and Schlmke (1966). The area under the peak for type I

(height x width at half-height) is about 7 times greater than the area

under the type II peak for brains from rats of both ages (figs. 4 and 5).

No type IV hexokinase was observed in either case.

The hexokinase isoenzymes eluted during chromatography of testis

extracts on the DEAE-cellulose column are illustrated in figs. 6-15

and sunmarized in table 2. In each case, 5 g of tissue were extracted,

so one should be able to compare quantities of each isoenzyme, if

differences in procedural losses are ignored.

Figures 6-8 demonstrate the developmental changes which occur in

hexokinase isoenzymes chromatographically isolated from rat testis ex­

tracts. Only types I and II were chromatographically found in testis extracts from 20-day old rats (fig. 6); unlike brain tissue, the testis contained nearly fourfold more type II hexokinase than type I. Total 29

Figure 4 . Chromatographic separation of brain hexokinase iso­

enzyme I (first peak) and II (second peak) from a

20-dsy-old rat. Extract from 12 g of brain was

applied to the DEAE cellulose column. Enzyme activ­

ity is represented by the solid line.

Figure 5. Chromatographic effluent after applying extracts of

5 g of adult rat brain to a DEAE-cellulose column.

See legend to figure 4 for description. 40i Fig. 4

30

2 0

•> 10

» 40

Fig. 5

2 0 -

10 20 Fraction Number (IOO ml) 31

Figures 6-9. Chromatographic profiles of hexokinase isoenzymes in

testes of normal rats of various ages. Types I and

XX are seen in figures 6 and 7, andtypes I, II and

IV are seen in figures 8 and 9. Each extract repre­

sents 5 g of testis tissue. Protein concentrations

estimated by absorption at 280 mu are represented by

the dashed line curves. See legend to fig. 4 for

description.

Fig. 6. Testes from 20-day-old rats.

Fig. 7. Testes from 30-day-old rats.

Fig. 8. Testes from 40-day-old rats.

Fig. 9. Testes from adult rats. 40i A O.D. (280 mil) (280 A O.D. uj 30

10 20 Fraction Number (10.0 ml) 40i A O.D. (280 mu) ENZYME ENZYME ACTIVITY (m|imoles/min/ml)

Fraction Number (10.0 ml) 34 quantities of the two testicular isoenzymes appeared to increase in

30-day old rats (fig. 7), hut the relative amounts of the two appeared unchanged. In 40-day old rats (fig, 8), types I and II are similar in level and ratio to the previous group; however, type IV isoenzyme

(glucokinase) was present in this chromatographic effluent. The extracts of adult rat testes resulted in a chromatographic profile (fig. 9) similar to, but slightly greater than, that seen for 40-day old rats; again, type II was the predominant form, but types I and IV were easily recog­ nizable .

When testes were taken from 20-day old rate which had been treated for 10 days with HCG, the effluent pattern depended on the HCG used

(compare figs. 10 and 11). When HCG from Ayerst Laboratories was used, only isoenzymes I and II were present. Compared to the normal 20-day old rat testis (fig. 6) however, type I was proportionally increased.

When HCG from Sigma Chemical Co. was used, however, an entirely different pattern emerged (fig. U ) . Hexokinases type I and II were present in actual and proportional amounts that were quite similar to those seen for young (20 or 30 day old) rats; in addition, however, a distinct glucoklnase (type IV) peak was found.

Hypophy sec tony had no apparent effects on isoenzymes I and II

(fig. 12). Type IV was not present, even though it would be expected in normal rats of this age and weight (see figs. 8 and 9). Treatment with HCG (Sigma) for 7 or 14 days (figs. 13 and 14) resulted in increased amounts of the isoenzymes present, but failed to promote the appearance of type IV (even though it did so in 20-day old rate). 35

Figures 10-15. Chromatographic separation of hexokinase isoenzymes

I, II and IV in extracts of 5 g of testis tissue from

experimentally altered and/or HCG-treated rats. See

legend to fig. 4 for description.

Fig. 10. Testes from 20-day-old rats treated for 10 days with

HCG (100 IU/rat/day; Ayerst Labs. Inc.)

Fig. 11. Testes from 20-day-old rats treated for 10 days with

HCG (100 IU/rat/day; Sigma Chemical Co.)

Fig. 12. Testes from rats hypophysectomized 3 weeks

Fig. 13. Testes from hypophysectomized rats treated for 7 days

with HCG (100 IU/rat/day; Sigma Chemical Co.)

Fig. 1 4 . Testes from hypophysectomized rats treated for 14 days

with HCG (100 IU/rat/day; Sigma Chemical Co.)

Fig. 15. Testes from rats cryptorchid for 4 weeks Rg. 10 30 •1.4

20 -

-0.8

-0.6 frtui 3 8 0 -0.2 ) a V 0

iii Fig.11

30- LlI -1.4

-1.2

20 - 1.0

-0.8

-0.6

■0.4

Fraction Number (10.0 ml) 40i

Fig. 12

- 1.6 3 0

■ 1.4

-1.2

-1.0

-0.6

-0.4

-0.2

40

Fig.13 A A (280 D. 0. mil)

30- ■1.4

2 0 ■ 1.0

0 6

-0.6 10- ■0.4

-02

10 20 30 Fraction Number (10.0 ml) Enzyme Activity (mjimoles/min/ml)

A O.D. (280 mu) 39 TABLE 2

SUMMARY OF HEXOKINASE ISOENZYMES IDENTIFIED

BY DEAE CELLULOSE CHROMATOGRAPHY

Group® Relative Concentration of Isoenzymes I II IV

Normal Rats

20 days old 2 3 0 30 days old 2 3 0 40 days old 2 3 1 Adult 2 3 1

HCG-treated Rats0

Ayerst - HCG 2 3 0 Sigma - HCG 2 3 1

Hypophysectomized Ratsd

HypoX controls 2 3 0 HCG - 7 days 2 “ 3 0 HCG - 14 days 2 3 0 .a 4-week Cryptorchid Rats 2 3 1

See text for details of treatments. b Subjectively rated on a quantitative scale of 0 (none present) to 3 (large amounts). No "sperm type" or type III isoenzyme was found In column assay. c 20 day old rats. d Adult rats. 40

Restricting testes of adult rats to the abdomen for A weeks did not alter the number of isoenzymes present (fig. 1$), compared to scrotal testes of the normal adult rat (fig. 9). However, the ratio of type I to type II increased in the cryptorchid tissue, apparently because of an increase in type I and a small decrease in type II.

Electrophoresis of Hexokinase Isoenzymes

In order to identify the hexokinase isoenzymes on polyacrylamide gels, rat brain extracts were used as a source of hexokinase type I and bull sperm were used to provide marker "sperm type” hexokinase. The former were prepared as described for column chromatography and the bull sperm extracts were prepared as described below.

Bull semen was collected using an artificial vagina and the sperm were separated from seminal plasma by centrifugation. The sperm were suspended in the Tris-Cl buffer to a dilution of 2x10^ cells/ml and sonicated for 5 minutes. The sample was frozen in liquid nitrogen and thawed three times, then centrifuged at 45,OCX) x g for 60 rain. The supernatant was collected for enzyme assay.

The patterns of isoenzymes found on electrophoretograms from the various experimental groups are diagrammatically shown in figure 16.

In each case, the relationship between types I and II was confirmed, though the predominance of the latter was not so striking. As well, a distinct, relatively wide band of glucokinase (IV) was present in the cases where a type IV peak was found; however, thinner bonds correspond­ ing to glucokinase were found for all other testis extracts. Type III isoenzyme, absent from all chromatographic profiles, was present in 41

Figure 16. Diagrammatic representation of hexokinase isoenzymes

separated by polyacrylamide disc gel electrophoresis

of experimental tissues. Fig. 16

ST

I

II

III IV

+ Normal Bull Rat 20-d 30* d 40-d Adult Sperm Brain Testis Testis Test) s Testis

ST i!ii *•«*< Ml*

MM 1 ill*, ■ M b in* ■ »*i

***■♦**** \XW ■ll** **•1 II !**«».•a** Ult ItM 41*1 >•**1

III !•••• i*M Mf* >41* • i|| IV •«** - **«• -

I 20-d 3 whs 3 whs 3 whs 4 whs + HCG Hypo* Hypo* Hypo* Testis Testis Safi 7-d HCG 14-d HCG restis- Testis Testis 43 I minor amounts in all samples electrophoresed.

The "sperm type" enzyme found In bull sperm was also found in all testis extracts except that from 20-day-old normal rats, whether sperm were present or not. Although it was absent from the brain extracts shown, and, in subsequent assays, from liver, skeletal muscle, heart and kidney, this isoenzyme was detected in occasional extracts from adult male rat brains and in seminal vesicles. DISCUSSION

Application of DEAE cellulose chromatography (Gonzalez e£ al.,

1964) and starch gel electrophoresis (Moore et al., 1964) techniques to the purification of hexokinase revealed that this enzymatic activity resided in a variable number of molecular forms, depending on the species and tissue. Since that time, a number of workers have confirmed and extended this finding.

Early work in this area showed that hexokinase type I far exceeded all other forms in rat brain; type II was present in low amounts and type

III was barely distinguishable (Grossbard and Schimke, 1966; Katzen et al..

1968). The findings in the present study confirm the earlier reports and extend them to the use of polyacrylamide gel electrophoresis (fig.

16). In no case has type IV isoenzyme (glucokinase) been isolated from brain tissue. Other tissues in which type I exceeds type II include the kidney, liver (Katzen, 1967; Grossbard and Schimke, 1966), and erythro­ cyte (Katzen, 1967), all of which are relatively insulin insensitive.

As shown earlier in this dissertation, hexokinase type II exceeds all other forms in the rat testis, regardless of age or treatment of rats (figs. 6-16). Katzen (1967), in his diagrammatic representation of enzyme levels, showed that types I and II were present in nearly equal amounts in rat testes and Pilkis et al. (1968), using a similar report­ ing technique, suggested that type I predominated in testes from rats, hamsters and gerbils. However, these workers based their measurements on staining intensity of starch gel electrophoretograms and the data reported here were collected using DEAE cellulose columns (figs. 6-15) or disc gel electrophoresis (fig. 16).

U 45

In those tissues which are highly sensitive to insulin, type II

appears to exceed type I (Katzen et al., 1968); fasting and diabetes both lead to a loss in type II which is overcome by refeeding or insulin treatment (Moore et al., 1964; Katzen, 1967). This appears true for diaphragm, skeletal muscle, heart and epididymal fat pad of young rats

(Katzen ei al., 1968). From the data reported here, it is also true of rat testes, which are also insulin sensitive.

In hypophysectamized rats, hexokinase II was not appreciably affected in testes (figs. 12 and 16), or liver (Pilkis, 1970), but this form was reduced to half in adipose tissue by hypophysectony (Pilkis,

1970) or thyroidectomy (Sharraa et al., 1963; Pilkis, 1970). Isoenzyme

II was slightly increased in testes by HCG treatment of hypophysectamized rats (figs. 14 and 16) and returned to normal in adipose tissue by thyroxine treatment of thyroidectomized rats (Pilkis, 1970). Thyroxine appears to enhance the activity of gonadotrophins on the testis (Massie,

1970), but the relationship of this hormone to testicular hexokinase is unknown.

Hexokinase type III is a major contributor to total enzyme levels in the liver, lung and spleen (Katzen ei el., 1968), but it is present in lesser amounts in other tissues and very low amounts in the testis

(fig. 16; Katzen, 1967; Pilkis et ill., 1968). Testicular levels of iso­ enzyme II appear unaffected by treatment or by the age of the animal.

Katzen (1967) and Katzen si al. (1968) reported that hexokinase IV, or glucokinase, waB limited to liver tissue, but the present study and other workers have clearly demonstrated the presence of this isoenzyme in testis tissue (figs. 8, 9, 11, 15, 16; Hansen et al., 1967; Pilkis et al.. 1968). Thi3 isoenzyme was not detected in chromatographic effluent from testis extracts of 20 or 30-day-old rats (figs. 6 and 7), but was found in small amounts when such extracts were subjected to disc gel electrophoresis (fig. 16), Rats treated with Sigma HCG for

10 days and sacrificed at 20 days of age, rats 40 days old or older, and cryptorchid rats all contained testicular glucoklnase in levels detect­ able by either technique. Hypophysectomy of rats resulted in testes with immature levels of glucokinase (i.e. detectable using electro­ phoresis, but not chromatography); this was not overcome by HCG treat­ ment.

Hypophysectomy reduced hepatic glucokinase (Sharma ei fll., 1963;

Pilkis, 1970), as did starvation or diabetes (Walker, 1966); this reduc­ tion was overcome by injections of hydrocortisone (Pilkis, 1970), but not thyroxine (Sharma, 1963). The changes which occurred in testicular hexokinase in the present study suggest that it may be related to stimulation of the testes by gonadotrophin. As shown with Ayerst HCG and with Sigma HCG administered to hypophysectomized rats, however, glucokinase apparently requires the action of endogenous gonadotrophin in conjunction with exogenous HCG. Sharma and Weinhouse (1963) were also unable to demonstrate HCG induction of glucokinase in hypophysec- tomized rats.

Katzen, in 1967, first reported a new isoenzyme of hexokinase in extracts of rat testes and epididymal fat pad. This isoenzyme, which migrated very slowly in the starch gel system used, was absent from the immature testis and from all other tissues studied, including adipose tissue from adult females. Therefore, Katzen attributed this form to sperm in the testes and to sperm contamination in the epididymal fat pad and named the isoenzyme "sperm type". Pilkis e± j|l. (1968) con­ firmed the presence of such an isoenzyme following starch gel electro­ phoresis of testis extracts from rats, hamsters and gerbils, but desig­ nated the form testis (T) type hexokinase, refusing to link it to a specific cell type. The results shown in figure 16 suggest that this isoenzyme is not linked to the sperm cell, since it was found in testes prior to the appearance of sperm (30-day-old) and in testes altered so that sperm were absent (hypophysectomized, eryptorchid). As well, the presence of the isoenzyme in other tissues in the adult male (epididymal fat pad, seminal vesicles, occasionally brain, and, according to Schimke and Grossbard in a 1968 note, perhaps liver), but not in the immature male or the female, suggests that this isoenzyme might be sensitive to testosterone levels, as is seminal vesicular hexokinase in general

(Singhal and Ling, 1969).

The "sperm type" of hexokinase was not observed when extracts were chromatographed on DEAE cellulose, because this isoenzyme was excluded in the initial column wash. SUMuUUff

Rat testis hexokinase isoenzymes were studied using DEAE cellulose

column chromatography and disc gel electrophoresis. Hexokinase iso­

enzymes I, XI, II, IV (glucokinase) and "sperm" or "testis" type

were measured in testes from growing and adult rats, immature,

hypophysectomized and HCG-treated rats, and cryptorehid rats.

"Sperm type" hexokinase was present in all testes except those from

20-day-old rats, whether sperm were present or not. It Is suggested

that this isoenzyme may be enhanced by testosterone in several

tissues.

Type II hexokinase exceeded type I in all testis preparations and was the predominant form in the testes. Although minor changes In

levels and relative amounts of these isoenzymes were found, no direct

relationship to testis function could be established.

Hexokinase type IV, glucokinase, was present in all testis extracts

applied to electrophoresis, but was below detectable limits in

immature or hypophysectomized rats. HCG increased the level of glucokinase in immature, but not hypophysectoinized rats. REFERENCES

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