003 1-399818912502-015 1 $02.00/0 PEDIATRIC RESEARCH Vol. 25, No. 2, 1989 Copyright O 1989 International Pediatric Research Foundation, Inc. Prinrc.d in (I.S. A. Galactose Metabolism in Human Ovarian Tissue Y-K. XU,' W. G. NG, F. R. KAUFMAN, R. A. LOBO, AND G. N. DONNELL Department of Pediatrics [Y.K.X., W.G.N., F.R.K., G.N. D.], Children's Hospital of Los Angeles, Los Angeles, Califi,rniu 90054, and Department of obstetric.^ and Gynecology, University of Southern California School of Medicine, Los Angeles Cozlnty/University of Sotlthern Califi~rniaWomen's Hospital, Los Angeles, California 90033 ABSTRACT. Galactose metabolism was studied in human treatment is begun in early infancy (1). The clinical symptoms ovarian tissue obtained from 14 women controls between of gonadal insufficiency have included partial or complete failure 21 and 72 y of age, and one 21-y-old galactosemic patient of secondary sexual development, primary amenorrhea, the post- with hypergonadotrophic hypogonadism. Tissue slices pubertal occurrence of oligomenorrhea, and secondary amenor- were incubated with l-'4C-galactose, and labeled interme- rhea. Although streak ovaries have been observed in two patients diates were analyzed by anion-exchange column chroma- (2, 3), the pathophysiology of the ovarian insult is unknown. It tography. Activities of enzymes related to the galactose has been hypothesized that metabolites of galactose, particularly pathway: galactokinase, transferase, epimerase, uridine di- galactose-I-phosphate, may be toxic to the ovarian parenchyma phosphoglucose (UDPGlc) and uridine diphosphogalactose during the prenatal state (4) or the perinatal period (1). The exact pyrophosphorylases, and UDPGlc and uridine diphospho- mechanism of this toxicity, however, has not been elucidated. galactose pyrophosphatases were measured in ovarian ho- To gain a better understanding of galactose metabolism in the mogenates using radioisotopic, spectrophotometric, and normal human ovary, galactose oxidation and its by-products fluorometric techniques. Incorporation of carbon label from were studied in ovarian tissue slices. In addition, the enzymes l-'4C-galactose into various galactose and glycolytic inter- involved in the metabolic process were assayed, including galac- mediates, as well as carbon dioxide and TCA-insoluble tokinase, galactose- 1-phosphate uridyl transferase, UDPGal-4- materials was demonstrated in samples from non-galacto- epimerase, UDPGal and UDPGlc pyrophosphorylases, and semic controls. In tissue from the galactosemic individual, UDPGal and UDPGlc pyrophosphatases. Preliminary results in no labeled carbon dioxide was produced and very little part have been presented (5). incorporation into TCA-insoluble material was found. La- beled galactose-1-phosphate was elevated. In normal ovar- ian tissue, specific activities of galactokinase, transferase, epimerase, and UDPGlc pyrophosphorylase are much MATERIALS AND METHODS higher than those found in the red cells and in testes. l-14C-~-galactose(1 5 mCi/mmol), and (U)-14C-a-~-galactose- UDPGlc pyrophosphorylase activity is about 50 times that 1-phosphate, potassium salt (8.2 mCi/mmol) were initially pur- of transferase, suggesting that uridine nucleotide sugars chased from Rosechem Products (Los Angeles, CA) and later have an important role in the normal development and from New England Nuclear Corp (Boston, MA). I-'4C-~-glucose function of the ovary. It is hypothesized that premature (5 mCi/mmol), UDP(U)-'4C-galactose (298.5 mCi/mmol) and ovarian failure. often observed in ~atientswith galactose- UDP(U)-14C-glucose(226.8 mCi/mmol) also were purchased mia, is due to interference with nucleotide sugar metabo- from New England Nuclear. lism and the synthesis of galactose containing glycoproteins Low EEO agarose (A-6013, type I), low gelling temperature and glycolipids consequent to the enzymatic defect in the agarose (A-40 18, type VII), UDPGlc, glucose- 1-phosphate, glu- major pathway of galactose metabolism. (Pediatr Res cose- l,6-diphosphate, adenosine-5 '-triphosphate, Triton X- 100. 25:151-155,1989) UTP, cysteine, galactose-1-phosphate and UDPGlc dehydrogen- ase were obtained from Sigma Chemical Co. (St. Louis, MO). Abbreviations NAD, 2(N-morpholino) ethane sulfonic acid, sodium salt mono- hydrate (Mes, ~a),and DTT were from Calbiochem (San Diego. transferase, galactose-1-phosphate uridyl transferase CA). G6PD and PGM were from BoehringerlMannheim (Indi- epimerase, uridine diphosphogalactose-4-epimerase anapolis, IN). DE 81 was from Whatman Co. (Clifton, NJ). G6PD, glucose-6-phosphate dehydrogenase Spectrafluor PPO/POPOP concentrate was from Amersham (Ar- PGM, phosphoglucomutase lington Heights, IL). Beta Blend was from West Chem Products TCA, trichloroacetic acid (San Diego, CA). UDPGlc, uridine diphosphoglucose Ovarian tissue was obtained from a 21-y-old galactosemic UDPGal, uridine diphosphogalactose patient with incipient ovarian failure during laparoscopy and UTP, uridine triphosphate from 14 nonealactosemic adults. between 21 and 72 v of age. undergoing tital abdominal hysterectomy and bilateral salprn- goophorectomy- - for uterine disease, breast cancer, and unilateral Premature ovarian failure has been found with high frequency ovarian cancer, in which case, based on histology, the nonpath- in female patients affected with galactosemia, even when dietary ologic ovary was used. Nine patients were premenopausal with normal ovarian function. and five were ~ostmeno~ausal.One Received April 19, 1988; accepted September 23, 1988. postmenopausal woman was receiving estrogen-progestin ther- Reprint requests W. G. Ng. Division of Medical Genetics, Children's Hospital apy at the time of oop~orectomy~~h~ postmenopausa~tissue of Los Angeles, P.O. Box 54700, Los Angeles, CA 90054. I Yan-Kang Xu is a research fellow in the Division of Medical Genetics, CHLA, was, aS expected, fibrotic and relatively devoid of follicles. from Sun-Yat Sen University of Medical Sciences. Guangzhou, China. Testicular tissue was obtained from men undergoing gonadec- 151 T AL. tomy for prostatic or testicular cancer, in which case the non- dioactive method similar to transferase except that UTP was pathologic gonad was studied. Informed consents were obtained used intead of UDPGlc and MgC12 (6.14 mM) was also added. before surgery. Tissues, once removed, were placed immediately 6) UDPGal pyrophosphatase activity was assayed by measur- in cold Krebs-Ringer bicarbonate glucose (KRB-glucose, 100 ing the amount of labeled galactose-I-phosphate formed from mg/100 ml) buffer, pH 7.4, before biochemical analysis. UDPGal during incubation (1 I). The incubation mixture (total Incubation studies. Ovarian tissue slices were prepared by vol, 50 p1) contained 2 (N-morpholino) ethane sulfonic acid hand, using sections of potato for grips. Each slice weighed buffer, pH 6.8, 123 mM; Triton X-100, 0.05%, UDP (I4C) Gal, approximately 1 mg. The slices were washed in KRB-glucose 0.14 mM, and 20 p1 tissue supernate (about 200 pg protein). At ( 100 mg/ 100 ml) buffer, pH 7.4 several times to free away any the end of 15 min at 37"C, the reaction was stopped by adding contaminated potato. Slices were incubated with either 1 pCi of 25 p1 of ethanol containing 5% acetic acid. After centrifugation galactose or 1 pCi of glucose in counting vials as previously (1000 x g, for 3 min), 25 p1 of supernate was spotted on DE 81 described (6). At the end of 2 h of incubation, labeled C02was paper and chromatographed as for transferase, UDPGal, galac- liberated upon addition of acid, collected, and counted. The tose-1-phosphate and galactose were separated. The amount of incubation mixture was subsequently treated with 10% TCA. UDPGal hydrolyzed is based on the sum of galactose-l-phos- The TCA-soluble and TCA-insoluble fractions were separated. phate, and galactose produced as tissue also contains hexose The TCA-insoluble fraction was then extracted with chloroform- phosphatase converting galactose-1-phosphate to galactose. methanol mixture (3: 1) for glycolipids, and the radioactivity was 7) UDPGlc pyrophosphatase activity was assayed by using an counted. The TCA in the TCA-soluble fraction was removed incubation mixture containing Tris buffer (pH 8.0), 400 mM; with ether, and the aqueous fraction was chromatographed on a I4C-UDPGlc, 0.14 mM; Triton X- 100, 0.05% and 20 11 tissue Dowex-1-formate column eluted with an ammonium formate extract (about 150-200 pg protein) in a total volume of 50 p1. gradient (7). At the end of 30 min of incubation, the reaction was stopped by Preparation of tissue homogenate for enzyme studies. Tissue heating at 100°C for 2 min. After centrifugation (10,000 x g, 3 samples of 200-400 mg were blotted with tissue paper and min), 20 p1 of supernate was spotted on DE 81 paper. The homogenized in 1.0 ml of a solution composed of 1 part of 0.1 chromatographic procedure and calculation for activity are sim- M DTT, 2 parts of 1.0 M glycine buffer, pH 8.7, and 7 parts of ilar to that of UDPGal pyrophosphatase. water in a tissue homogenizer (Kontes Co., Vineland, NJ) in an Protein concentration was determined by the method of Lowry ice water bath for 5 min. The preparation was then centrifuged et al. (12) with BSA as standard. UDPGlc and UDPGal levels at 12,000 x g for 15 min at 4"C, and the supernatant was used were determined by modified methods of Fujimura et al. (13). for enzyme assays and protein determinations. Enzyme assays. All enzyme activities were expressed in terms RESULTS of products formed, pmollhlg protein. Each enzyme assay has been standardized for linearity in the rate of product formation Table I compares the results of I4CO2 production and of the and concentrations of protein used. incorporation of carbon label into TCA-insoluble material from 1)Galactokinase activity was determined by a modification of labeled galactose and glucose in the ovarian tissue slices from the method of Ng et al. (8). The incubation mixture contained the galactosemic patient and nongalactosemic controls. With Tris buffer (pH 7.0), 100 mM; MgSO,, 40 mM; NaF, 5 mM; galactose as the substrate, no labeled C02was produced by the ATP, 4 mM; 14C-galactose,0.555 mM and 50 p1 tissue extract tissue from the patient with galactosemia.