Indi an Journal of Experimental Biology Vol. 38, November 2000, pp. 1104-1110

SDS-PAGE analysis of caput epididymis proteins in rats receiving a zinc deficient diet

Ranjana Agrawal & R S Bedwal*

Cell Biology Laboratory, Department of Zoology, Uni versity of Rajasthan, Jaipur 302 004

Received 29 May / 998; revised 3 August 2000

Capu t epid idymi s proteins fro m contro l, pairfed and zi nc deficient (ZD) wistar weanling albi no rats after 2-, 4-, 6- and 8-wccks were examined using SDS-PAGE fo llowed by densitometric scanning of the gels. In compari son to the control and pairfed rats, ZD rats di splayed new protei ns. These included a Mr 42 kDa from 2ZD, Mr 47.5, 27.5, 23.2 and 16.0 kDa from 4ZD and Mr 87 and 14.2 kDa fro m 6ZD group. The 8ZD group, however, revealed no additi onal protein bands over con­tro ls. Further, several other proteins were mi ssing from ZD rats. These included Mr 93 and 7 1 kDa from 2ZD; 93 , 90, 79, 67, 62, 55 and 15.3 kDa from 4ZD; 60, 45.5, 34, 30 and 24 kDa from 6ZD and 4 1.5, 33 and 27.5 kDa bands from 8ZD group. The results indicate that the induced Zn-defi cient state may be responsible for the altered protein patterns in the caput epididymis . The duration of low Zn uptake period also appears to influence the protein pattern in caput epid idymis.

Zinc, an essenti al biological trace element, exhibits 60% hi gher uptake (as 65Zn) into adult rat epididymis as compared to testes'. Localization of zinc in the epididymis at ul trastructural leve l revealed its pres­ence in the ves icles and in the lysosome- like struc­tures of apical part of the principal cell s while the luminal grains were associated with sperm ce ll s, with the surface of large microv illi (stereoc ili a) or free in the seminal fluids of epididymisv. Stain ing with the element-specific fluorophore, zinquin has revealed bright fluorescence over the entire head of testicul ar spermatozoa. However comparati vely more bright flu orescence than in post-acrosomal region of sper­matozoa from caput epididymis was observed than over the head of spermatozoa from the cauda epi­didymis of mouse4

. Thus different ia l di stributi on of zinquin flu orescence and its loss by treatment of spermatozoa with Triton X-1 00, suggests that Zn is assoc iated with plasma membrane and is possi bl y involved in maturation of spermatozoa during passage th rough ep ididymis2

.4. In vitro experiments on human spermatozoa indicate that one of the functions of zinc is to preserve sperm nuclear chromatin decondensa­lion ·(NCO) ability till the appropriate stage of male genome transfer and thereby play a crucial role in fertili zati on5

·6

. Moreover, as and rogen receptor has two zinc fin gers, Zn is probab ly in vo lved in the modul ati on of androgen regul ated gene ex pression7

*Correspondent au tho r.

The secretions of epididymis, that contribute to maturati onal changes in spermatozoa, are largely proteinous in nature. And according to protein syn­thesis index values, caput epididymi s synthesizes more proteins in vivo than cauda epididymis8

. The present study has been des igned to examine the ef­fects of Zn deficiency on the protein pro fil e of caput epididymis fo ll owing I 0 -gel electrophoresis and den­sitometric scanning.

Materials and Methods Wistar albino rats of 40 days of age and weighing

around I OOg body weight were selected from a co lony of inbred Wistar rats and grouped at random into following 3 groups of I 0 animals each :

I. Control group (ZC): The animals were fed on a synthetic diet having I 00 ppm zinc.

2. Pairfed group (PF): The ani mals were fed on I 00 ppm zinc in synthetic diet but the amount given was equal to that consumed by zinc deficient group.

3. Zinc defi cient group (ZD): The animals were fed on a synthetic diet with < I ppm zinc.

The basal diet was prepared according to Wall ace et a/.9. Briefl y, the diet compri sed (%) corn starch, 7.5; dex trose, 50; egg white fl akes/ powder, 20; corn oil , I 0; salt mi xture, 3.7 [K2HPO". 1.4; CaC03, 0.99; NaCI, 0.55; MgSO", 0.38; Ca2HPO.~. 0.3 1; ferric citrate, 0.09; KI, 0.002; CuS04 , 0.0009; MnS04 ,

0.0008; CoCb, 0.000 I] ; Vitamin - anti bioti c mixture, 2.2 (sucrose, 1.76; choline chl oride, 0.37; ampicillin ,

AGRAWAL & BEDWAL: CAPUT EPIDIDYMIS PROTEINS IN Zn DEFICIENT RATS 1105

0.024; Vitamin-E-acetate, 0.022; Vitamin D2, 0.0033 ; Vitamin A, 0.0026; calcium pentothenate, 0.0015; thiamine HCI, 0 .0009; riboflavin, 0.0006; biotin, 0.0004; pyridoxin HCI, 0.0004; niacin, 0.0002; folic acid, 0 .00008; monadione, 0 .00008; cobalamine, 0.00001 ); calcium carbonate, 2; DL - methionein, I; phytic acid, 1 and cellulose, 5. Zinc contents of diet (after digestion in diacidic solution, HN03 : HCI04 ::

5: I) from each lot were estimated on a GBC-902 double beam atomic absorption spectrophotometer in air acetylene flame at 213.9 nm, 0 .5 nm slit width, 3mA hollow cathode lamp current, with background correction and single integration with an integration time of 3 sec. Zinc concentration was adjusted to Jess than I ppm for zinc deficient diet and I 00 ppm for control diet by adding appropriate amount of zinc sulphate. The cages, grills and bottles were washed with tap water and twice with demineralized water and then with I 0% EDT A solution to prevent any contamination with Zn. The animals were autopsied under mild anaesthesia after 2-, 4-, 6- and 8-weeks and the caput epididymis excised, cleared of extraneous tissues, weighed on electronic balance and processed for SDS-PAGE analysis. Briefly, caput epididymis (I 00 mg/ml) from each group (ZC, ZD, PF) were homogenized in 0.25 mM sucrose solution, centrifuged at I 0,000 rpm at 4°C for I 0 min and the supernatant was denatured by sodium dodecyl sulphate (SDS) and mercaptoethanol. The denatured proteins (70 ~g, estimated by Bradford's method) were then loaded into wells of a slab gel discontinuous SDS-PAGels [17.5 em (L) x 15 em (B) x 1.5 em (T)] prepared by the method of Laemmli 10

,

along with 2% bromophenol blue as tracking dye . Separating (resolving) gel of 11 % (pH 8.8) and stacking gel of 3.5% (pH 6.8) were used . Standard protein markers (a-lactalbumin 14.2 kDa, trypsin inhibitor 20.1 kDa, trypsinogen 24 kDa, carbonic anhydrase 36 kDa, egg albumin 45 kDa and bovine albumin 66 kDa) were also run along with tissue samples. The electrophoresis was carried out in Tris­Glycine buffer (pH8.3) for 4 hrs, initially at 20 rnA but as the tracking dye bromophenol blue approached the junction of stacking and separating gels, the current was doubled. The gels were fixed in a methanol, acetic acid and distilled water (4: I :5) fixative, stained by the method of Dzandu et al. 11 and were then subjected to semiquantitative estimation at

610 nm on Quick Scan Densitometer (DESAGA gmBH). Approximate number of amino acid residues in a protein were calculated by dividing its relative molecular weight by 110.

Although the average molecular weight of 20 standard amino acids is about 138, the smaller amino acids predominant in most proteins, when weighted for the proportions in which the various amino acids occur in proteins, the average molecular weight is 128. Further, since a molecule of water (Mr, 18) is eliminated to form a peptide bond, the average

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Fig. 1-SDS-PAGE profile (a) and densitometric scanning (b) of standard weight marker proteins ( 14.2 kDa, a-lactalbumin; 20. 1 kDa, trypsin inhibitor; 24 kDa, trypsinogen; 29 kDa, carbonic anhydrase; 36 kDa, glyceraldehyde phosphate dehydrogenase; 45 kDa, egg albumin and 66 kDa, bovine albumin).

0 01

Table !-Protein profiles (Mr) and no. of amino acid residues per protein molecule in caput epididymis of zinc deficient weanling albino rats

Protein profiles (M r) No. of amino Protein profiles (Mr) No. of amino Protein profiles (Mr) No. of amino Protein profiles (M r) No. of amino (kDa) acid residues (kDa) acid residues (kDa) acid residues (kDa) acid residues

2ZC 2PF 2ZD per protein 4ZC 4PF 4ZD per protein 6ZC 6PF 6ZD per protein 8ZC 8PF 8ZD per protein

93 - 845.4545 93 - 845 .4545 91 91 91 827.2727 87 87 87 790.9090 77 77 77 700.00 90 - - 818.18 18 - - 87 790.9090 82 82 82 745.4545

71 - . 645.4545 87 87 87 790.9090 - 76 76 690.9090 77.5 77.5 77.5 704.5454 68 68 68 6 18. 18 18 - 85 85 772.7272 73 73 73 663.6363 74 74 74 672.7272 64 64 64 581.8181 79 79 - 718.1818 70 70 70 636.3636 71 7 1 71 645.4545 60 60 60 545.4545 77 77 77 700.00 66 66 66 600.00 69 69 69 627.2727 z 58 58 58 527.2727 74 74 74 672.7272 - 62 62 563.6363 65 65 65 590.9090 0 56 56 56 509.0909 71 7 1 71 645.4545 60 - 545.4545 61 61 61 554.5454 ;;

z 53 53 53 481.8181 67 - - 609.0909 57 57 57 518.1818 55 55 55 500.00 '-

50 50 50 454.5454 64 64 581.8181 54 54 54 490.9090 51 51 51 463.6363 tTl X

47.5 47.5 47.5 431.8181 62 - - 563.6363 51 - 5 1 463.6363 46 46 46 418.1818 "'0

44 44 44 400.00 60 60 545.4545 48.5 48.5 48.5 440.9090 45 45 45 409.0909 t:C

- 0 42 381.8 181 55 - - 500.00 45.5 - - 413.6363 41.5 41.5 - 377.2727 r

39.5 39.5 . 359.0909 52 52 52 472.7272 - 44 44 400.00 39.5 39.5 39.5 359.0909 z 36.5 36.5 36.5 331.8181 49.5 49.5 450.00 40.5 40.5 40.5 368.1818 38 38 38 345.4545

0 - <

35 35 38 1.1 8 18 47.5 431.8181 38 38 38 345.4545 35 35 35 318.181 8 tTl - - 3::: 33 33 33 300.00 45.5 45 .5 45.5 413.6363 - 36 36 327.2727 33 - - 300.00 o:l

30.5 30.5 tTl

30.5 277.2727 43 - 43 390.9090 34 - - 309.0909 - 32 32 290.9090 ;>;:l

28.5 28.5 28 .5 259.0909 40.5 40.5 40.5 368. 18 18 33 33 33 300.00 29 29 29 263 .6363 N 0

26.5 26.5 26.5 240.9090 38 38 38 345.4545 31 - 31 281.8181 27.5 250.00 0

- - 0

24 24 24 218 .1 8 18 34.5 34.5 34.5 3 13.6363 - 30 - 272.7272 - 26.5 26.5 240.9090 21 21 21 190.9090 31.5 31.5 31.5 286.3636 28.5 28.5 28 .5 259.0909 25.5 25.5 25.5 23 1.8181 20. 20 20 181.8 181 29.5 29.5 29.5 268. 1818 27.5 - 27.5 250.00 23 23 23 209.0909 19 19 19 172.7272 - - 27.5 250.00 24.0 - - 218.1818 17.5 17.5 17.5 159.0909 18 18 18 163.6363 25 25 25 227.2727 - 23.5 23.5 213.6363 14.2 14.2 14.2 129.0909 17 17 17 154.5454 - - 23.2 210.9090 17 .5 17.5 17.5 159.0909 15.5 15 .5 15.5 140.9090 18.5 - 18.5 168.1818 - - 14.2 129.0909 14 14 14 127.2727 - 18.0 18.0 163.6363

16.0 145.4545 15.3 - 139.0909

14.2 14.2 129.0909

AGRAWAL & BEDWAL : CAPUT EPIDIDYMIS PROTEINS IN Zn DEFICIENT RATS 1107

2ZC

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Fig. 2-Silver-Coomassie stai ned SDS-PAGE ( II %) gels illus­trating electrophoretic separation (a) and densitometric scanning (b) of caput epididymal proteins from 2-week experiment. In 2(a) lane 8,9 and I 0 for 2ZC, lane 5,6 and 7 for 2ZD and lane I ,2,3 and 4 for 2PF groups.

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Fig. 3-Silver-Coomassie stained SDS-PAGE ( II %) gels showi ng electrophoretic separation (a) and densitometric scanning (b) . of caput epididymal protei ns from 4-weeks ex periment. In 3(a) lane 2 and 3 for 4ZC, lane I ,4 and 5 for 4ZD and lane 6,7 and 8 for 4PF groups.

1108 INDIAN J EXP BIOL, NOVEMBER 2000

6ZC

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Fig. 4-Silver-Coomassie stained SDS-PAGE ( II %) gels exhib­iting electrophoretic separation (a) and densitometric scanning (b) of caput epididymal proteins from 6-wecks experiment. In 4(a) lane 4 for 6ZC, lane I and 3 for 6ZD and lane 2 for 6PF groups.

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Fig. 5-Sil vcr-Coomassie stained SDS-PAGE ( I I%) gels di s­playing electrophoretic separation (a) and den irometric scann ing (b) of caput epididymal proteins from 8-weeks experiment. In 5(a) lane 7,8 and 9 fo r 8ZC, lane 4,5 and 6 for 8ZD and lane I ,2 and 3

for 8PF groups.

AGRAWAL & BEDWAL: CAPUT EPIDIDYMIS PROTEINS IN Zn DEFICIENT RATS 1109

molecu lar weight of an amino ac id res idue is 128-1 8 =110 (Lehninger et al. 12

) .

Results and Discussion The results are presented in Table I and Figs. 1-5. Alterations in the protein profiles patterns in the

caput epididymis were as a result of zinc deficiency induced though a zinc deficient diet. Caput epididymis is a very active region in prote in synthes is and secretion and many of the reproductive and somatic genes such as HE2, B/C ( 18-19 kDa), EAP (89 kDa), GPX (24 kDa), CRES ( 14 kDa), enk (28 kDa), CRBP ( 14 kDa), A-raf (72 kDa) , Sa-reductase (28 kDa) , POMC (29-32 kDa), GGT (7 12 kDa), SGP-2 (701 kDa) , D/E (29-32 kDa) etc are expressed in fairly high concentrat ion 13

• SDS-PAGE protein pattern anal ysis of caput epididymis revealed new proteins in zinc defic ient animals e.g., I (Mr 42.0 kDa) from 2ZD, 4 (Mr 47 .5 , 27.5, 23.2 and 16.0 kDa) from 4ZD, 2 (Mr 87 and 14.2 kDa) from 6ZD and none from 8ZD group were new and hence absent in thei r respecti ve control and pairfed animals. In addition , 2 protei ns of Mr 79 kDa from 4-weeks and Mr 41 .5 kDa from 8-weeks caput epididymis were missing only from deficient groups as compared to thei r respect ive cont ro l and pai rfed groups. Similarly, several other proteins which could not be identified were 6 (Mr 93 .0, 90.0, 67.0, 62.0, 55.0 and 15.3 kDa) from 4ZD and 4PF, 4 (Mr 60.0, 45 .5, 34 and 24.0 kDa) from 6ZD and 6PF and 2 (Mr 33.0 and 27.5 kDa) in 8ZD and 8PF groups as compared to thei r respecti ve control groups. Futher, the proteins 3 (Mr 47 .5, 36.5 and 33.0 kDa) from 2ZD, 7 (Mr 52.0, 34.5, 3 1.5, 29.5 25.0, 18.5 and 18.0 kDa) from 4ZD, 4 (Mr 66.0, 51.0, 28.5 and 17.5 kDa) from 6ZD and 4 (Mr 61.0, 29.0, 23 .0 and 17 .5 kDa) from 8ZD decreased in ZD groups as against their respective controls. Reduced concentration and complete loss of some of the proteins may be on account of protein degradati on as a result of increased lysosomal enzymatic activity 14

• Caput epididymal epithelial cells, exposed to higher leve ls of androgens {primarily Sa-dihydro­testosterone (DHT) } through the peripheral circulation and directly from the testicul ar fluid , are the major factors involved in controlling the activities of epithelial cells and in determining the composition of epididymal luminal fluids 15

.

Recently , zinc has been implicated to influence maturation of spermatozoa during the ir passage through epididymis2

·16

, probably by binding to thi ol

groups of prote ins and the irby protecting protei n oxidation 17

• Chelation of zinc ions, on the other hand, has been shown to affect spe rm moti lit / 8

·19

. Zinc prevents programmed cell death or apoptosis whereas . h I . . d . t4 20 21 A . . tts c e atton tn uces tt · · . poptosts ts an energy-dependent process requmng de novo protein synthesis22

'21

• The dying cells express large amounts of mRNA for several degenerati ve enzymes and regulatory proteins maintain control over the apoptotic cascade24

. Zinc deficiency, therefore, may have led to altered prote in patterns in caput epididymis so as to avoid deteri mental effects. In addition, comparison of prote in profi les of ZD and PF group illustrated only few variations where as comparison of protein profil es of ZD and PF groups with their respective ZC groups ex hibited many variations. Thi s is probably as a result of reduced feed intake. It is concluded that induced zinc deficiency state may be responsible for the altered response in protein pattern analyzed from the caput epididymis.

Acknowledgement One of the author (Dr. Ranjana Agrawal) thanks

CSIR, New Delhi for award of Research Associateship and financial assistance.

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4 Zalewski P D, Ji an X, Soon L L L, Breed W G, Seamark R F, Lincoln S F, Ward A D & Sun F Z, Changes in distribu­ti on of labi le zinc in mouse spermatozoa during mat uration in the epididymis assessed by the flu orophore zinquin . Re­prod Ferlil Dev, 8 (1996) 1097.

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1110 INDIAN J EXP BIOL, NOVEMBER 2000

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19 Danscher G & Rebbe H, Effects of two chel ating agents, oxine and diethyldithiocarbamate (Antabuse), on stainabi lity and motility of human sperms. J Histochem Cytochem, 22 (1974) 98 1.

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